Coelenterazine analogs and manufacturing method thereof

ABSTRACT

There has been a need for coelenterazine analogs that exhibit luminescence properties different from those of known coelenterazine analogs. The present invention provides the compound represented by general formula (1).

TECHNICAL FIELD

The present invention relates to coelenterazine analogs, a method formanufacturing the same, and so on.

BACKGROUND ART

The phenomenon of bioluminescence was observed in some living species,and was based on the chemical reaction of a luciferin (a luminescencesubstrate) and a luciferase (an enzyme catalyzes the luminescencereaction) in vivo. A number of researches including the researches foridentification of luciferin and luciferase and for elucidation of theluminesence mechanism in a molecular level have been performed insideand outside of Japan.

In recent years, bioluminescence is used as a tool for biologicalresearch. In addition, the applied researches in the medical fieldincluding high through-put screening (HTS) of drugs, intramolecularimaging, etc., have been intensively developed on the basis of theprinciple of bioluminescence.

Fireflies, sea pansies Renilla, sea fireflies Cypridina, deep-seashrimps Oplophorus, luminescent microorganisms, etc. are known asrepresentative bioluminescent organisms that produce bioluminescence.The jellyfish Aequorea victoria is also a bioluminescent animal, but thebioluminescence of the jellyfish is not produced by the luciferasereaction. The luminescence is produced by the Ca²⁺-triggered reaction ofthe photoprotein of aequorin, the complex of substrate-enzyme-molecularoxygen. It is known that many organisms utilize the compound having animidazopyrazinone skeleton as a luminescence substrate in thebioluminescence system.

Among them, coelenterazine (CTZ) is a compound commonly used as aluminescence substrate (luciferin) for aequorin which is a photoproteinfrom jellyfish, or for luciferases from some bioluminescent organismssuch as sea pansies Renilla, etc. Therefore, many findings of CTZ havebeen accumulated.

In fact, approximately 50 types of coelenterazine analog (CTZ analog)have been synthesized heretofore, and the substrate specificity for someof them has been examined in several bioluminescence systems (cf., e.g.,Non-Patent Literatures 1 to 5).

CITATION LIST Non-Patent Literature

-   [Non-Patent Literature 1] Shimomura O. et al., Biochem. J. 251,    405-410 (1988)-   [Non-Patent Literature 2] Shimomura O. et al., Biochem. J. 261,    913-920 (1989)-   [Non-Patent Literature 3] Inouye S. & Shimomura O., Biochem.    Biophys. Res. Commun. 233, 349-353 (1997)-   [Non-Patent Literature 4] Inouye S. & Sasaki S., Protein Express.    Purif. 56, 261-268 (2007)-   [Non-Patent Literature 5] Inouye S. & Sahara Y., Biochem. Biophys.    Res. Commun. 265, 96-101 (2008)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

Under the foregoing situations, coelenterazine analogs having differentluminescence properties from those of known coelenterazine analogs havebeen sought.

Means of Solving the Problems

The present inventors have conducted extensive investigations to solvethe foregoing problems. As a result, the inventors have found thatcoelenterazine analogs having methyl, trifluoromethyl, methoxy or ethylin place of hydroxy on the benzene ring at the position 2 ofcoelenterazine possess luminescence properties, which are different fromthose of known coelenterazine analogs, and have come to attain thepresent invention.

That is, the present invention provides coelenterazine analogs, methodsfor producing coelenterazine analogs, and so on, which are shown below.

(1) A compound represented by general formula (1) below:

wherein:

R¹ is hydrogen, hydroxy, an alkyl having 1 to 4 carbon atoms which mayoptionally be substituted with an alicyclic group, trifluoromethyl or analkoxyl;

R² is hydrogen, hydroxy, a halogen, an alkyl having 1 to 4 carbon atomswhich may optionally be substituted with an alicyclic group,trifluoromethyl or an alkoxyl;

R³ is hydrogen, an alkyl having 1 to 4 carbon atoms which may optionallybe substituted with an alicyclic group, or an alkoxyl;

R⁴ is a substituted or unsubstituted aryl, a substituted orunsubstituted arylalkyl, a substituted or unsubstituted arylalkenyl, analkyl which may optionally be substituted with an alicyclic group, analkenyl which may optionally be substituted with an alicyclic group, analicyclic group, or a heterocyclic group;

R⁵ is hydrogen or a substituted or unsubstituted alkyl;

X¹ is hydrogen, hydroxy, a halogen, an alkoxyl or amino; and,

X² is hydrogen or hydroxy;

with the proviso that when R² and R³ are hydrogen, R¹ is an alkyl having1 to 4 carbon atoms which may optionally be substituted with analicyclic group, trifluoromethyl, or an alkoxyl,

when R¹ and R³ are hydrogen, R² is hydroxy, an alkyl having 1 to 4carbon atoms which may optionally be substituted with an alicyclicgroup, trifluoromethyl, or an alkoxyl, or,

when R¹ and R² are hydrogen, R³ is an alkyl having 1 to 4 carbon atomswhich may optionally be substituted with an alicyclic group, or analkoxyl.

(2) The compound according to (1) above, wherein R¹ is hydrogen,hydroxy, methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, trifluoromethyl, methoxy, ethoxy,n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy ortert-butoxy, in the general formula (1).

(3) The compound according to (1) or (2) above, wherein R² is hydrogen,hydroxy, fluorine, methyl, ethyl, propyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, trifluoromethyl,methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy,iso-butoxy, sec-butoxy or tert-butoxy, in the general formula (1).

(4) The compound according to any one of (1) to (3) above, wherein R³ ishydrogen, methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, methoxy, ethoxy, n-propoxy,iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy ortert-butoxy, in the general formula (1).

(5) The compound according to any one of (1) to (4) above, wherein R⁴ isphenyl, p-hydroxyphenyl, benzyl, α-hydroxybenzyl, phenylethyl,phenylvinyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl, methyl,ethyl, propyl, 2-methylpropyl, 2-methylpropenyl, adamantylmethyl,cyclopentylmethyl or thiophen-2-yl, in the general formula (1).

(6) The compound according to any one of (1) to (5) above, wherein, inthe general formula (1):

R¹ is hydrogen, methyl, ethyl, trifluoromethyl or methoxy;R² is hydrogen, hydroxy, methyl or methoxy;R³ is hydrogen or methyl;R⁴ is benzyl;R⁵ is hydrogen;X¹ is hydroxy; and,X² is hydrogen;

with the proviso that when R² and R³ are hydrogen, R¹ is methyl, ethyl,trifluoromethyl or methoxy,

when R¹ and R³ are hydrogen, R² is hydroxy, methyl or methoxy, or,

when R¹ and R² are hydrogen, R³ is methyl.

(7) The compound according to (6) above, which is represented by formulabelow.

(8) The compound according to (6) above, which is represented by formulabelow.

(9) The compound according to (6) above, which is represented by formulabelow.

(10) The compound according to (6) above, which is represented byformula below.

(11) The compound according to (6) above, which is represented byformula below.

(12) The compound according to (6) above, which is represented byformula below.

(13) The compound according to (6) above, which is represented byformula below.

(14) The compound according to (6) above, which is represented byformula below.

(15) A process for producing a compound represented by general formula(1) below:

wherein R¹, R², R³, R⁴, R⁵, X¹ and X² are the same as defined below,which comprises reacting a compound represented by general formula (2)below:

wherein:

R⁴ is a substituted or unsubstituted aryl, a substituted orunsubstituted arylalkyl, a substituted or unsubstituted arylalkenyl, analkyl which may optionally be substituted with an alicyclic group, analkenyl which may optionally be substituted with an alicyclic group, analicyclic group, or a heterocyclic group:

R⁵ is hydrogen or a substituted or unsubstituted alkyl;

X¹ is hydrogen, hydroxy, a halogen, an alkoxyl or amino; and,

X² is hydrogen or hydroxy;

with a compound represented by general formula (3) below:

wherein:

R¹ is hydrogen, hydroxy, an alkyl having 1 to 4 carbon atoms which mayoptionally be substituted with an alicyclic group, trifluoromethyl or analkoxyl;

R² is hydrogen, hydroxy, a halogen, an alkyl having 1 to 4 carbon atomswhich may optionally be substituted with an alicyclic group,trifluoromethyl or an alkoxyl; and,

R³ is hydrogen, an alkyl having 1 to 4 carbon atoms which may optionallybe substituted with an alicyclic group, or an alkoxy;

with the proviso that when R² and R³ are hydrogen, R¹ is an alkyl having1 to 4 carbon atoms which may optionally be substituted with analicyclic group, trifluoromethyl, or an alkoxyl,

when R¹ and R³ are hydrogen, R² is hydroxy, an alkyl having 1 to 4carbon atoms which may optionally be substituted with an alicyclicgroup, trifluoromethyl, or an alkoxyl, and,

when R¹ and R² are hydrogen, R³ is an alkyl having 1 to 4 carbon atomswhich may optionally be substituted with an alicyclic group, or analkoxyl.

(16) The process according to (15) above, wherein R¹ is hydrogen,hydroxy, methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, trifluoromethyl, methoxy, ethoxy,n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy ortert-butoxy, in the general formula (1).

(17) The process according to (15) or (16) above, wherein R² ishydrogen, hydroxy, fluorine, methyl, ethyl, propyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, trifluoromethyl,methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy,iso-butoxy, sec-butoxy or tert-butoxy, in the general formula (1).

(18) The process according to any one of (15) to (17) above, wherein R³is hydrogen, methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, methoxy, ethoxy, n-propoxy,iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy ortert-butoxy, in the general formula (1).

(19) The process according to any one of (15) to (18) above, wherein R⁴is phenyl, p-hydroxyphenyl, benzyl, α-hydroxybenzyl, phenylethyl,phenylvinyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl, methyl,ethyl, propyl, 2-methylpropyl, 2-methylpropenyl, adamantylmethyl,cyclopentylmethyl or thiophen-2-yl, in the general formula (1).

(20) The process according to any one of (15) to (19) above, wherein, inthe general formula (1):

R¹ is hydrogen, methyl, ethyl, trifluoromethyl or methoxy,R² is hydrogen, hydroxy, methyl or methoxy,R³ is hydrogen or methyl,R⁴ is benzyl,R⁵ is hydrogen,X¹ is hydroxy, and,X² is hydrogen,

with the proviso that when R² and R³ are hydrogen, R¹ is methyl, ethyl,trifluoromethyl or methoxy,

when R¹ and R³ are hydrogen, R² is hydroxy, methyl or methoxy, and,

when R¹ and R² are hydrogen, R³ is methyl.

(21) A method for producing a calcium-binding photoprotein, whichcomprises contacting the compound according to any one of (1) to (14)above with an apoprotein of the calcium-binding photoprotein to obtainthe calcium-binding photoprotein.

(22) A method for detecting or quantifying a calcium ion, whichcomprises using the calcium-binding photoprotein produced by the methodaccording to (21) above.

(23) A method for analyzing a physiological function or enzyme activity,which comprises performing a bioluminescence resonance energy transfer(BRET) assay using as a donor protein the calcium-binding photoproteinproduced by the method according to (21) above.

(24) A method for measuring a transcription activity or detecting ananalyte, which comprises using the compound according to any one of (1)to (14) above, and a luciferase derived from Renilla sp., Oplophorus sp.or Gaussia sp.

(25) A method for analyzing a physiological function or enzyme activity,which comprises performing a bioluminescence resonance energy transfer(BRET) assay using the compound according to any one of (1) to (14)above and a luciferase derived from Renilla sp., Oplophorus sp. orGaussia sp. as a donor protein.

(26) The method according to (24) or (25) above, wherein Renilla sp. isRenilla reniformis.

(27) The method according to (26) above, wherein the luciferase derivedfrom Renilla reniformis comprises a polypeptide consisting of the aminoacid sequence of SEQ ID NO: 14.

(28) The method according to (24) or (25) above, wherein Oplophorus sp.is Oplophorus gracilorostris.

(29) The method according to (28) above, wherein the luciferase derivedfrom Oplophorus gracilorostris comprises a polypeptide consisting of theamino acid sequence of SEQ ID NO: 16.

(30) The method according to (24) or (25) above, wherein Gaussia sp. isGaussia princeps.

(31) The method according to (30) above, wherein the luciferase derivedfrom Gaussia princeps comprises a polypeptide consisting of the aminoacid sequence of SEQ ID NO: 18.

(32) A kit for measuring a transcription activity or detecting ananalyte, comprising the compound according to any one of (1) to (14)above and a luciferase derived from at least one organism selected fromthe group consisting of Renilla sp., Oplophorus sp. and Gaussia sp.

(33) A kit for analyzing a physiological function or enzyme activity,comprising the compound according to any one of (1) to (14) above, aluciferase derived from at least one organism selected from the groupconsisting of Renilla sp., Oplophorus sp. and Gaussia sp. and at leastone selected from the group consisting of an organic compound and afluorescent protein, utilizing the principle of intermolecularinteraction by a bioluminescence resonance energy transfer (BRET) assay.

Effect of the Invention

The present invention provides novel coelenterazine analogs.Coelenterazine analogs in a preferred embodiment of the presentinvention exhibit the luminescence properties which are different fromthose of known coelenterazine analogs.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the relationship between the regeneration time andluminescence intensity of semi-synthetic aequorins.

FIG. 2 shows the luminescence patterns for semi-synthetic aequorins.

FIG. 3 shows the emission spectra of semi-synthetic aequorins byaddition of calcium ions.

FIG. 4 shows the relationship between the initial luminescence intensityof semi-synthetic aequorins and calcium ion levels.

FIG. 5 shows the emission spectra of Oplophorus luciferase by additionof coelenterazine or its analogs.

FIG. 6 shows the emission spectra of Gaussia luciferase by addition ofcoelenterazine or its analogs.

FIG. 7 shows the emission spectra of Renilla luciferase by addition ofcoelenterazine or its analogs.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail.

1. Coelenterazine Analog of the Invention

The present invention provides the following compound (coelenterazineanalog of the present invention) represented by general formula (1)below.

wherein:

R¹ is hydrogen, hydroxy, an alkyl having 1 to 4 carbon atoms which mayoptionally be substituted with an alicyclic group, trifluoromethyl or analkoxyl;

R² is hydrogen, hydroxy, a halogen, an alkyl having 1 to 4 carbon atomswhich may optionally be substituted with an alicyclic group,trifluoromethyl or an alkoxyl;

R³ is hydrogen, an alkyl having 1 to 4 carbon atoms which may optionallybe substituted with an alicyclic group, or an alkoxyl;

R⁴ is a substituted or unsubstituted aryl, a substituted orunsubstituted arylalkyl, a substituted or unsubstituted arylalkenyl, analkyl which may optionally be substituted with an alicyclic group, analkenyl which may optionally be substituted with an alicyclic group, analicyclic group, or a heterocyclic group;

R⁵ is hydrogen or a substituted or unsubstituted alkyl;

X¹ is hydrogen, hydroxy, a halogen, an alkoxyl or amino; and,

X² is hydrogen or hydroxy;

with the proviso that when R² and R³ are hydrogen, R¹ is an alkyl having1 to 4 carbon atoms which may optionally be substituted with analicyclic group, trifluoromethyl, or an alkoxyl;

when R¹ and R³ are hydrogen, R² is hydroxy, an alkyl having 1 to 4carbon atoms which may optionally be substituted with an alicyclicgroup, trifluoromethyl, or an alkoxyl; and,

when R¹ and R² are hydrogen, R³ is an alkyl having 1 to 4 carbon atomswhich may optionally be substituted with an alicyclic group, or analkoxyl.

The “alkyl having 1 to 4 carbon atoms which may optionally besubstituted with an alicyclic group” in R¹ is, for example, anunsubstituted straight or branched alkyl having 1 to 4 carbon atoms, ora straight or branched alkyl having 1 to 4 carbon atoms which issubstituted with, e.g., 1 to 10 alicyclic groups. Examples of thealicyclic group include cyclohexyl, cyclopentyl, adamantyl, cyclobutyl,cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl,cyclopentyl, adamantyl, etc. The “alkyl which may optionally besubstituted with an alicyclic group” is, for example, methyl, ethyl,propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, cyclobutylmethyl, cyclopropylmethyl,etc., preferably, methyl, ethyl, propyl, 2-methylpropyl,adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl,and the like. In some embodiments of the present invention, the “alkylwhich may optionally be substituted with an alicyclic group” is astraight alkyl which may optionally be substituted with an alicyclicgroup and examples are methyl, ethyl, propyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc.

The “alkoxyl” in R¹ is, for example, a straight or branched alkoxyhaving 1 to 6 carbon atoms. Examples of the “alkoxy” are methoxy,ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy,sec-butoxy, tert-butoxy, n-pentyloxy, iso-pentyloxy, sec-pentyloxy,1,1-dimethylpropyloxy, 1,2-dimethylpropoxy, 2,2-dimethylpropyloxy,n-hexoxy, 1-ethylpropoxy, 2-ethylpropoxy, 1-methylbutoxy,2-methylbutoxy, iso-hexoxy, 1-methyl-2-ethylpropoxy,1-ethyl-2-methylpropoxy, 1,1,2-trimethylpropoxy, 1,1,2-trimethylpropoxy,1-propylpropoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy,2,2-dimethylbutoxy, 2,3-dimethylbutyloxy, 1,3-dimethylbutyloxy,2-ethylbutoxy, 1,3-dimethylbutoxy, 2-methylpentoxy, 3-methylpentoxy,hexyloxy, etc. In some embodiments of the invention, the “alkoxy” ismethoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy,iso-butoxy, sec-butoxy or tert-butoxy, preferably, methoxy.

In a preferred embodiment of the invention, R¹ is hydrogen, hydroxy,methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, trifluoromethyl, methoxy, ethoxy,n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy ortert-butoxy. In a more preferred embodiment of the invention, R¹ ishydrogen, methyl, ethyl, trifluoromethyl or methoxy.

The “halogen” in R² is, for example, fluorine, chlorine, bromine oriodine. In a preferred embodiment of the present invention, the“halogen” is fluorine.

The “alkyl having 1 to 4 carbon atoms which may optionally besubstituted with an alicyclic group” in R² is, for example, anunsubstituted straight or branched alkyl having 1 to 4 carbon atoms, ora straight or branched alkyl having 1 to 4 carbon atoms which issubstituted with, e.g., 1 to 10 alicyclic groups. Examples of thealicyclic group include cyclohexyl, cyclopentyl, adamantyl, cyclobutyl,cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl,cyclopentyl, adamantyl, etc. The “alkyl which may optionally besubstituted with an alicyclic group” is, for example, methyl, ethyl,propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, cyclobutylmethyl, cyclopropylmethyl,etc., preferably, methyl, ethyl, propyl, 2-methylpropyl,adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl,etc. In some embodiments of the present invention, the “alkyl which mayoptionally be substituted with an alicyclic group” is a straight alkylwhich may optionally be substituted with an alicyclic group and examplesare methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, etc.

The “alkoxyl” in R² is, for example, a straight or branched alkoxyhaving 1 to 6 carbon atoms, and examples are methoxy, ethoxy, n-propoxy,iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,n-pentyloxy, iso-pentyloxy, sec-pentyloxy, 1,1-dimethylpropyloxy,1,2-dimethylpropoxy, 2,2-dimethylpropyloxy, n-hexoxy, 1-ethylpropoxy,2-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy, iso-hexoxy,1-methyl-2-ethylpropoxy, 1-ethyl-2-methylpropoxy,1,1,2-trimethylpropoxy, 1,1,2-trimethylpropoxy, 1-propylpropoxy,1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 2,2-dimethylbutoxy,2,3-dimethylbutyloxy, 1,3-dimethylbutyloxy, 2-ethylbutoxy,1,3-dimethylbutoxy, 2-methylpentoxy, 3-methylpentoxy, hexyloxy, etc. Insome embodiments of the invention, the “alkoxy” is methoxy, ethoxy,n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy ortert-butoxy, preferably, methoxy.

In a preferred embodiment of the invention, R² is hydrogen, hydroxy,fluorine, methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, trifluoromethyl, methoxy, ethoxy,n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy ortert-butoxy. In a more preferred embodiment of the invention, R² ishydrogen, hydroxy, methyl or methoxy.

The “alkyl having 1 to 4 carbon atoms which may optionally besubstituted with an alicyclic group” in R³ is, for example, anunsubstituted straight or branched alkyl having 1 to 4 carbon atoms, ora straight or branched alkyl having 1 to 4 carbon atoms which issubstituted with, e.g., 1 to 10 alicyclic groups. Examples of thealicyclic group include cyclohexyl, cyclopentyl, adamantyl, cyclobutyl,cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl,cyclopentyl, adamantyl, etc. The “alkyl which may optionally besubstituted with an alicyclic group” is, for example, methyl, ethyl,propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, cyclobutylmethyl, cyclopropylmethyl,etc., preferably, methyl, ethyl, propyl, 2-methylpropyl,adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl,etc. In some embodiments of the invention, the “alkyl which mayoptionally be substituted with an alicyclic group” is a straight alkylwhich may optionally be substituted with an alicyclic group, andexamples include methyl, ethyl, propyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc.

The “alkoxyl” in R³ is, for example, a straight or branched alkoxyhaving 1 to 6 carbon atoms. Examples of the “alkoxy” are methoxy,ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy,sec-butoxy, tert-butoxy, n-pentyloxy, iso-pentyloxy, sec-pentyloxy,1,1-dimethylpropyloxy, 1,2-dimethylpropoxy, 2,2-dimethylpropyloxy,n-hexoxy, 1-ethylpropoxy, 2-ethylpropoxy, 1-methylbutoxy,2-methylbutoxy, iso-hexoxy, 1-methyl-2-ethylpropoxy,1-ethyl-2-methylpropoxy, 1,1,2-trimethylpropoxy, 1,1,2-trimethylpropoxy,1-propylpropoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy,2,2-dimethylbutoxy, 2,3-dimethylbutyloxy, 1,3-dimethylbutyloxy,2-ethylbutoxy, 1,3-dimethylbutoxy, 2-methylpentoxy, 3-methylpentoxy,hexyloxy, etc. In some embodiments of the invention, the “alkoxy” ismethoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy,iso-butoxy, sec-butoxy or tert-butoxy, preferably, methoxy.

In a preferred embodiment of the invention, R³ is hydrogen, methyl,ethyl, propyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl,cyclohexylethyl, methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy,n-butoxy, iso-butoxy, sec-butoxy or tert-butoxy. In a more preferredembodiment of the invention, R³ is hydrogen or methyl.

The “substituted or unsubstituted aryl” in R⁴ is, for example, an arylhaving 1 to 5 substituents or an unsubstituted aryl. The substituent is,for example, at least one selected from the group consisting of ahalogen (fluorine, chlorine, bromine or iodine, etc.), hydroxy, an alkylhaving 1 to 6 carbon atoms, an alkoxyl having 1 to 6 carbon atoms, aminoand a dialkylamino having 1 to 6 carbon atoms. In some embodiments ofthe invention, the substituent is hydroxy. Specific examples of the“substituted or unsubstituted aryl” are phenyl, p-hydroxyphenyl,p-aminophenyl, p-dimethylaminophenyl, etc., preferably, phenyl,p-hydroxyphenyl, etc. In some embodiments of the invention, the“substituted or unsubstituted aryl” is an unsubstituted aryl, e.g.,phenyl, etc.

The “substituted or unsubstituted arylalkyl” in R⁴ is, for example, anarylalkyl having 7 to 10 carbon atoms, which is substituted with 1 to 5substituents, or an unsubstituted arylalkyl having 7 to 10 carbon atoms.The substituent includes, for example, a halogen (fluorine, chlorine,bromine or iodine, etc.), hydroxy, an alkyl having 1 to 6 carbon atoms,an alkoxyl having 1 to 6 carbon atoms, amino, a dialkylamino having 1 to6 carbon atoms, etc. Examples of the “substituted or unsubstitutedarylalkyl” are benzyl, α-hydroxybenzyl, phenylethyl, p-hydroxybenzyl,p-dimethylaminobenzyl, etc., preferably, benzyl, α-hydroxybenzyl,phenylethyl, etc. In some embodiments of the invention, the “substitutedor unsubstituted arylalkyl” is benzyl.

The “substituted or unsubstituted arylalkenyl” in R⁴ is, for example, anarylalkenyl of 8 to 10 carbon atoms having 1 to 5 substituents, or anunsubstituted arylalkenyl having 8 to 10 carbon atoms. The substituentincludes, for example, a halogen (fluorine, chlorine, bromine or iodine,etc.), hydroxy, an alkyl having 1 to 6 carbon atoms, an alkoxyl having 1to 6 carbon atoms, amino, a dialkylamino having 1 to 6 carbon atoms,etc. Examples of the “substituted or unsubstituted arylalkenyl” arephenylvinyl, p-hydroxyphenylvinyl, p-dimethylaminophenylvinyl, etc. Insome embodiments of the present invention, the “substituted orunsubstituted arylalkenyl” is an unsubstituted arylalkenyl, e.g.,phenylvinyl, etc.

The “alkyl which may optionally be substituted with an alicyclic group”in R⁴ is, for example, an unsubstituted straight or branched alkylhaving 1 to 4 carbon atoms, or a straight or branched alkyl having 1 to4 carbon atoms which is substituted with, e.g., 1 to 10 alicyclicgroups. Examples of the alicyclic group include cyclohexyl, cyclopentyl,adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, the alicyclic groupis cyclohexyl, cyclopentyl, adamantyl, etc. Examples of the “alkyl whichmay optionally be substituted with an alicyclic group” are methyl,ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, cyclobutylmethyl, cyclopropylmethyl,etc., preferably, methyl, ethyl, propyl, 2-methylpropyl,adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl,etc. In some embodiments of the present invention, the “alkyl which mayoptionally be substituted with an alicyclic group” is a straight alkylwhich may optionally be substituted with an alicyclic group, andexamples include methyl, ethyl, propyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc.

The “alkenyl which may optionally be substituted with an alicyclicgroup” in R⁴ is, for example, an unsubstituted straight or branchedalkenyl having 2 to 6 carbon atoms, or a straight or branched alkenylhaving 2 to 6 carbon atoms which is substituted with, e.g., 1 to 10alicyclic groups. Examples of the alicyclic group include cyclohexyl,cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, thealicyclic group is cyclohexyl, cyclopentyl, adamantyl, etc. Examples ofthe “alkenyl which may optionally be substituted with an alicyclicgroup” include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl,3-butenyl, 2-methylpropenyl, etc., preferably, 2-methylpropenyl, etc.

The “alicyclic group” in R⁴ includes, for example, cyclohexyl,cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, thealicyclic group is cyclohexyl, etc.

The “heterocyclic group” in R⁴ includes, for example, a group derivedfrom a 5- to 7-membered ring containing, in addition to carbon atoms, 1to 3 atoms selected from the group consisting of N, O and S as the atomsconstituting the ring and bonded via carbon atoms, a group formed byfusing 2 or more of such rings and bonded via carbon, or a group formedby fusing such a ring to a benzene ring and bonding via carbon atoms.Examples of the “heterocyclic group” are thiophen-2-yl, 2-furanyl,4-pyridyl, etc. In some embodiments of the present invention, the“heterocyclic group” is a heterocyclic group containing sulfur, e.g.,thiophen-2-yl.

In a preferred embodiment of the present invention. R⁴ is phenyl,p-hydroxyphenyl, benzyl, α-hydroxybenzyl, phenylethyl, phenylvinyl,cyclohexyl, cyclohexylmethyl, cyclohexylethyl, methyl, ethyl, propyl,2-methylpropyl, 2-methylpropenyl, adamantylmethyl, cyclopentylmethyl orthiophen-2-yl. In a more preferred embodiment of the present invention,R⁴ is benzyl.

The “substituted or unsubstituted alkyl” in R⁵ is, for example, an alkylof 1 to 6 carbon atoms having, e.g., 1 to 6 substituents, or anunsubstituted alkyl having 1 to 6 carbon atoms. The substituent is, forexample, at least one selected from the group consisting of a halogen(fluorine, chlorine, bromine or iodine, etc.), hydroxy, carboxyl, analkyl having 1 to 6 carbon atoms, an alkoxyl having 1 to 6 carbon atoms,amino and a dialkylamino having 1 to 6 carbon atoms. In some embodimentsof the invention, the substituent is hydroxy. Specific examples of the“substituted or unsubstituted alkyl” are methyl, 2-hydroxyethyl,carboxymethyl, 3-hydroxypropyl, etc., preferably, methyl,2-hydroxyethyl, etc.

In a preferred embodiment of the present invention, R⁵ is hydrogen,methyl or 2-hydroxyethyl. In a more preferred embodiment of the presentinvention, R⁵ is hydrogen.

The “halogen” in X¹ is, for example, fluorine, chlorine, bromine oriodine. In a preferred embodiment of the present invention, the“halogen” is fluorine.

The “alkoxyl” in X¹ is an alkoxyl having, e.g., 1 to 6 carbon atoms, andexamples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropoxy,tert-butyloxy, etc. In a preferred embodiment of the present invention,the “alkoxyl” is methoxy.

In a preferred embodiment of the present invention, X¹ is hydrogen,hydroxy, fluorine, methoxy or amino. In a more preferred embodiment ofthe present invention, X¹ is hydroxy.

In a preferred embodiment of the present invention, X² is hydrogen.

In some embodiments of the present invention, the following symbolsrepresent as follows in the general formula (1):

R¹ is hydrogen, methyl, ethyl, trifluoromethyl or methoxy;R² is hydrogen, hydroxy, methyl or methoxy;R³ is hydrogen or methyl;R⁴ is benzyl;R⁵ is hydrogen;X¹ is hydroxy; and,X² is hydrogen;

with the proviso that when R² and R³ are hydrogen, R¹ is methyl, ethyl,trifluoromethyl or methoxy;

when R¹ and R³ are hydrogen, R² is hydroxy, methyl or methoxy; and,

when R¹ and R² are hydrogen, R³ is methyl.

In an embodiment of the present invention, the compound represented bygeneral formula (1) is a compound represented by the formula below.

In another embodiment of the present invention, the compound representedby general formula (1) is a compound represented by the formula below.

In a still another embodiment of the present invention, the compoundrepresented by general formula (1) is a compound represented by theformula below.

In a still another embodiment of the present invention, the compoundrepresented by general formula (1) is a compound represented by theformula below.

In a still another embodiment of the present invention, the compoundrepresented by general formula (1) is a compound represented by theformula below.

In a still another embodiment of the present invention, the compoundrepresented by general formula (1) is a compound represented by theformula below.

In a still another embodiment of the present invention, the compoundrepresented by general formula (1) is a compound represented by theformula below.

In a still another embodiment of the present invention, the compoundrepresented by general formula (1) is a compound represented by theformula below.

Coelenterazine analogs in an embodiment of the present invention exhibitthe luminescence properties, which are different from those of knowncoelenterazine analogs (e.g., h-coelenterazine, n-coelenterazine,i-coelenterazine, etc.). Coelenterazine analogs in some embodiments ofthe present invention become relatively good luminescence substrates forat least one luciferase selected from the group consisting of Oplophorusluciferase, Renilla luciferase and Gaussia luciferase. Coelenterazineanalogs in a preferred embodiment of the present invention becomerelatively good luminescence substrates for Oplophorus luciferase,Renilla luciferase and Gaussia luciferase.

2. Process for Producing Coelenterazine Analog of the Invention

The compound represented by general formula (1) (coelenterazine analogof the invention) can be produced as follows.

That is, the compound represented by general formula (1) can be producedby reacting the compound represented by general formula (2) below:

(wherein R⁴, R⁵, X¹ and X² are as defined above) with the compoundrepresented by general formula (3) below:

(wherein R¹, R² and R³ are as defined above), whereby the compoundrepresented by general formula (1) can be obtained.

The compound represented by general formula (2) can be prepared by knownprocesses. For example, the compound represented by general formula (2)can be prepared, e.g., by the process described in Kishi, Y. et al.,Tetrahedron Lett., 13, 2747-2748 (1972), or Adamezyk, M. et al., Org.Prep. Proced. Int., 33, 477-485 (2001), or their modifications. Morespecifically, the compound represented by general formula (2) can beprepared as follows. That is, first, cyclization of a substitutedphenylglyoxal aldoxime and a glycinonitrile derivative is carried outusing a Lewis acid catalyst to form the pyrazine oxide. Subsequently,the pyrazine oxide is subjected to catalytic hydrogenation using RaneyNi, etc. as a catalyst to prepare the compound. Alternatively, thecompound represented by general formula (2) can be prepared byconducting the Suzuki-Miyaura coupling reaction between a2-amino-5-bromopyrazine derivative and a substituted phenylboronic acidpinacol ester.

The compound represented by general formula (3) can be prepared by knownprocesses. For example, the compound represented by general formula (3)can be prepared, e.g., by the processes described in Adamczyk, M. etal., Synth. Commun., 32, 3199-3205 (2002), or Baganz, H. & May, H.-J.Chem. Ber., 99, 3766-3770 (1966) and Baganz, H. & May, H.-J. Angew.Chem., Int. Ed. Eng., 5, 420 (1966), or their modifications. Morespecifically, the compound represented by general formula (3) can beprepared as follows. That is, the compound represented by generalformula (3) can be prepared either by reacting a substituted benzylGrignard reagent with ethyl diethoxyacetate at a low temperature (−78°C.), or by reacting an α-diazo-α′-substituted phenyl ketone withtert-butyl hypochlorite in ethanol.

Herein, the solvent used for the process for producing the compound ofthe present invention represented by general formula (1) is notparticularly limited and various solvents can be used. Examples of thesolvent include dioxane, tetrahydrofuran, ether, methanol, ethanol,water, etc. These solvents can be used alone or as an admixture thereof.

In the process of producing the compound of the present inventionrepresented by general formula (1), the reaction temperature andreaction time are not particularly limited and are, for example, 0° C.to 200° C. for 1 to 96 hours, room temperature to 150° C. for 3 to 72hours, or 60° C. to 120° C. for 6 to 24 hours.

3. Method for Producing Calcium-Binding Photoprotein

The calcium-binding photoprotein of the invention can be produced orregenerated by contacting the compound represented by general formula(1) (coelenterazine analog of the invention) with the apoprotein of thecalcium-binding photoprotein thereby to obtain the calcium-bindingphotoprotein.

As used herein, the term “contact” means that coelenterazine analog ofthe invention and the apoprotein of the calcium-binding photoprotein areallowed to be present in the same reaction system, and includes, forexample, the apoprotein of the calcium-binding photoprotein being addedto a container charged with coelenterazine analog of the invention,coelenterazine analog of the invention being added to a containercharged with the apoprotein of the calcium-binding photoprotein,coelenterazine analog of the invention being mixed with the apoproteinof the calcium-binding photoprotein, and the like. In one embodiment ofthe present invention, the contact is carried out at a low temperaturein the presence of a reducing agent (e.g., mercaptoethanol,dithiothreitol, etc.) and oxygen. More specifically, the photoprotein ofthe present invention can be produced or regenerated by the methodsdescribed in, e.g., Shimomura, O. et al. Biochem. J. 251, 405-410(1988), Shimomura, O. et al. Biochem. J. 261, 913-920 (1989), and thelike. The calcium-binding photoprotein of the present invention ispresent in such a state that a complex is formed between the peroxide ofcoelenterazine analog generated from coelenterazine analog of theinvention and molecular oxygen and the apoprotein. Calcium ions arebound to the complex above to generate instantaneous luminescence andform coelenteramide analog, which is the oxide of coelenterazine analog,and carbon dioxide. The complex above is sometimes referred to as “thephotoprotein of the present invention.”

The apoprotein used to produce the photoprotein of the present inventionincludes, for example, apoaequorin, apoclytin-I, apoclytin-II, apobelin,apomitrocomin, apomineopsin, apobervoin, and the like. In someembodiments of the present invention, the apoprotein is apoaequorin,apobelin, apoclytin-I, apoclytin-II, mitrocomin, etc., e.g.,apoaequorin. These apoproteins may be obtained from natural sources orgenetically engineered. Furthermore, the amino acid sequence may bemutated from the natural sequence by gene recombination technology, solong as the apoproteins are capable of producing the calcium-bindingphotoprotein.

The nucleotide sequences and amino acid sequences of the apoproteins ofphotoproteins obtained from the nature (natural apoproteins) are asfollows. That is, the nucleotide sequence and amino acid sequence ofnatural apoaequorin are represented by SEQ ID NO: 1 and SEQ ID NO: 2.The nucleotide sequence and amino acid sequence of natural apoclytin-Iare represented by SEQ ID NO: 3 and SEQ ID NO: 4. The nucleotidesequence and amino acid sequence of natural apoclytin-II are representedby SEQ ID NO: 5 and SEQ ID NO: 6. The nucleotide sequence and amino acidsequence of natural apomitrocomin are represented by SEQ ID NO: 7 andSEQ ID NO: 8. The nucleotide sequence and amino acid sequence of naturalapobelin are represented by SEQ ID NO: 9 and SEQ ID NO: 10. Thenucleotide sequence and amino acid sequence of natural apobervoin arerepresented by SEQ ID NO: 11 and SEQ ID NO: 12.

The apoprotein mutated by recombinant technology is a protein selectedfrom the group consisting of (a) to (c) below:

(a) a protein comprising the amino acid sequence of natural apoproteinin which 1 or more amino acids are deleted, substituted, inserted and/oradded, and having the apoprotein activity or function of thecalcium-binding photoprotein;

(b) a protein comprising an amino acid sequence which is 90% or morehomologous to the amino acid sequence of natural apoprotein, and havingthe apoprotein activity or function of the calcium-binding photoprotein;and,

(c) a protein comprising an amino acid sequence encoded by apolynucleotide that hybridizes under stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of natural apoprotein, and having the apoproteinactivity or function of the calcium-binding photoprotein.

Examples of the “natural apoprotein” described above are apoaequorin,apoclytin-I, apoclytin-II, apobelin, apomitrocomin, apomineopsin,apobervoin, etc. In an embodiment of the present invention, theapoprotein is apoaequorin, apoclytin-I, apoclytin-II, apobelin,apomitrocomin, etc., preferably apoaequorin. The amino acid sequencesand nucleotide sequences of these natural apoproteins are as describedabove.

The “apoprotein activity or function of the calcium-bindingphotoprotein” means the activity or function that, e.g., the apoproteinbinds to the peroxide of coelenterazine or the peroxide of acoelenterazine analog to produce the calcium-binding photoprotein.Specifically, “the protein binds to the peroxide of coelenterazine orthe peroxide of a coelenterazine analog to produce the calcium-bindingphotoprotein” not only means that (1) the protein binds to the peroxideof coelenterazine or the peroxide of a coelenterazine analog to producethe photoprotein, but also means that (2) the protein is brought intocontact with coelenterazine or its derivative in the presence of oxygento produce a photoprotein (complex) comprising the protein and theperoxide of coelenterazine or the peroxide of a coelenterazine analog.As used herein, the term “contact” means that the protein andcoelenterazine or its analog are allowed to be present in the samereaction system, and includes, for example, the protein being added to acontainer charged with coelenterazine or its analog, coelenterazine orits analog being added to a container charged with the protein, theprotein being mixed with coelenterazine or its analog, and the like. The“coelenterazine analog” refers to a compound which is capable ofconstituting a calcium-binding photoprotein such as aequorin, etc.,together with the apoprotein in the same manner as in coelenterazine.Examples of coelenterazine or its analog include, in addition tocoelenterazine analogs of the present invention, coelenterazine,h-coelenterazine, f-coelenterazine, cl-coelenterazine, n-coelenterazine,cp-coelenterazine, ch-coelenterazine, hch-coelenterazine,fch-coelenterazine, e-coelenterazine, ef-coelenterazine,ech-coelenterazine, hcp-coelenterazine, and the like. Coelenterazineanalogs of the present invention can be produced, e.g., by the processesdescribed above or their modifications. The other coelenterazines ortheir analogs can be produced by the processes described in, e.g.,Shimomura et al. (1988) Biochem. J. 251, 405-410, Shimomura et al.(1989) Biochem. J., 261, 913-920, Shimomura et al. (1990) Biochem. J.,270, 309-312, or their modifications. Alternatively, various types ofcoelenterazine analogs are commercially available from ChissoCorporation, Wako Pure Chemical Industry Co., Ltd. and Promega Inc., andthese commercial products may also be used.

The range of “1 or more” in “the amino acid sequence in which 1 or moreamino acids are deleted, substituted, inserted and/or added” describedabove is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1. Ingeneral, the less the number of amino acids deleted, substituted,inserted or added, the more preferable. In the deletion, substitution,insertion and addition of the amino acid residues described above, twoor more may occur concurrently. Such regions can be acquired usingsite-directed mutagenesis described in “Sambrook J. et al., MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press (2001),” “Ausbel F. M. et al., Current Protocols inMolecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997),”“Nuc. Acids. Res., 10, 6487 (1982),” “Proc. Natl. Acad. Sci. USA, 79,6409 (1982),” “Gene, 34, 315 (1985),” “Nuc. Acids. Res., 13, 4431(1985),” “Proc. Natl. Acad. Sci. USA, 82, 488 (1985),” etc.

The range of “90% or more” in the “amino acid sequence which is 90% ormore homologous” described above is, for example, 90% or more, 91% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more,99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% ormore, 99.8% or more, or 99.9% or more. It is generally preferred for thenumerical value indicating the degree of homology to be higher. Thehomology between amino acid sequences or nucleotide sequences can bedetermined using a sequencing program such as BLAST (see, e.g.,Altzchul, S. F. et al., J. Mol. Biol., 215, 403 (1990), etc.) or thelike. When BLAST is used, the default parameters for the respectiveprograms are employed.

The “polynucleotide that hybridizes under stringent conditions”described above refers to a polynucleotide (e.g., DNA) which is obtainedby, for example, colony hybridization, plaque hybridization or Southernhybridization using as the probe a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of naturalapoprotein or all or part of the polynucleotide encoding the amino acidsequence of natural apoprotein. Specific examples include apolynucleotide which can be identified by performing hybridization at65° C. in the presence of 0.7 to 1.0 mol/L NaCl using a filter on whichthe polynucleotide from the colony or plaque is immobilized, thenwashing the filter at 65° C. with 0.1- to 2-fold SSC (saline-sodiumcitrate) solution (a 1-fold SSC solution is composed of 150 mmol/l,sodium chloride and 15 mmol/L sodium citrate).

Hybridization may be performed in accordance with modifications of themethods described in manuals, e.g., Sambrook, J. et al.: MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press (2001), Ausbel F. M. et al., Current Protocols inMolecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997),Glover D. M. and Hames B. D., DNA Cloning 1: Core Techniques, Apractical Approach, Second Edition, Oxford University Press (1995), etc.

As used herein, “stringent conditions” may refer to less stringentconditions, moderately stringent conditions and highly stringentconditions. The “less stringent conditions” are, for example, theconditions under 5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS and 50%(v/v) formamide at 32° C. The “moderately stringent conditions” are, forexample, the conditions under 5×SSC, 5×Denhardt's solution, 0.5% (w/v)SDS and 50% (v/v) formamide at 42° C. The “highly stringent conditions”are, for example, the conditions under 5×SSC, 5×Denhardt's solution,0.5% (w/v) SDS and 50% (v/v) formamide at 50° C. The more stringent theconditions are, the higher the complementarity required for doublestrand formation. Specifically, for example, under these conditions, apolynucleotide (e.g., DNA) of higher homology is expected to be obtainedefficiently at higher temperatures, although multiple factors areinvolved in hybridization stringency, including temperature, probeconcentration, probe length, ionic strength, time and saltconcentration; those skilled in the art may appropriately choose thesefactors to realize a similar stringency.

Where a kit commercially available is used for the hybridization, forexample, Alkphos Direct Labeling Reagents (manufactured by AmershamPharmacia) may be used. In this case, incubation with a labeled probe isperformed overnight in accordance with the protocol attached to the kit,the membrane is then washed with a primary wash buffer containing 0.1%(w/v) SDS at 55° C., and finally the hybridized DNA can be detected.

Other hybridizable polynucleotides include, as calculated by asequencing program such as BLAST or the like using the defaultparameters, DNAs having a homology of approximately 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more,90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% ormore, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, or99.9% or more, to the polynucleotide encoding the amino acid sequence ofthe apoprotein. The homology of amino acid sequences or nucleotidesequences can be determined using the method described above.

The recombinant apoprotein which can be used in the present inventionincludes, for example, recombinant aequorin described in Shimomura, O.and Inouye, S. Protein Express. Purif. (1999) 16: 91-95, recombinantclytin-I described in Inouye, S. and Sahara, Y. Protein Express. Purif.(2007) 53: 384-389, recombinant clytin-II described in Inouye, S. J.Biochem. (2008) 143: 711-717, and the like.

The calcium-binding photoprotein thus obtained may be further purified.Purification of the calcium-binding photoprotein may be performed in aconventional manner of separation/purification. Theseparation/purification includes, for example, precipitation withammonium sulfate, gel filtration chromatography, ion exchangechromatography, affinity chromatography, reversed phase high performanceliquid chromatography, dialysis, ultrafiltration, etc., alone or in anappropriate combination thereof.

The photoprotein in an embodiment of the present invention exhibits theluminescence properties which are different from those of knownphotoproteins. The photoprotein in some embodiments of the presentinvention show less Ca²⁺ sensitivity when compared to the photoproteincontaining natural coelenterazine, and are well suited for applicationsto a high-precision assay system in which Ca²⁺ level change in thesystem is used as an indicator, in the same manner as in thephotoprotein containing i-CTZ or n-CTZ.

4. Application of Coelenterazine Analog of the Invention or thePhotoprotein of the Invention (1) Use as Luminescence Substrate

Coelenterazine analog in some embodiments of the present invention emitslight by the action of a luminescent catalyst and can thus be used as aluminescence substrate. Accordingly, the present invention provides alight-emitting method, which comprises contacting coelenterazine analogof the present invention with a luminescent catalyst. As used herein,the term “contact” means that coelenterazine analog of the invention andthe luminescent catalyst are allowed to be present in the same reactionsystem, and includes, for example, the luminescent catalyst being addedto a container charged with coelenterazine analog, coelenterazine analogbeing added to a container charged with the luminescent catalyst,coelenterazine analog being mixed with the luminescent catalyst, and thelike.

The luminescent catalyst used for the light-emitting method of thepresent invention includes, for example, a luciferase derived fromOplophorus sp. (e.g., Oplophorus gracilorostris) (Oplophorusluciferase), a luciferase derived from Gaussia sp. (e.g., Gaussiaprinceps) (Gaussia luciferase), a luciferase derived from Renilla sp.(e.g., Renilla reniformis or Renilla muelleri) (Renilla luciferase), aluciferase derived from Pleuromamma sp. (Pleuromamma luciferase), or aluciferase derived from Metridia longa (Metridia luciferase).Coelenterazine analog in some embodiments of the present invention actsas a luminescence substrate for Oplophorus luciferase, Gaussialuciferase or Renilla luciferase. Coelenterazine analog in an embodimentof the present invention acts as a luminescence substrate for Oplophorusluciferase. Coelenterazine analog in another embodiment of the presentinvention acts as a luminescence substrate for Oplophorus luciferase,Gaussia luciferase and Renilla luciferase. These luminescent catalystscan be produced by the method described in, e.g., Shimomura et al.(1988) Biochem. J. 251, 405-410, Shimomura et al. (1989) Biochem. J.261, 913-920, or Shimomura et al. (1990) Biochem. J. 270, 309-312, ormodifications thereof. Alternatively, various products are commerciallyavailable from Chisso Corporation, Wako Pure Chemical Industry Co., Ltd.and Promega Inc., and these commercial products may also be used for thelight-emitting method of the present invention.

Now, of Renilla luciferase, the nucleotide sequence and amino acidsequence of the luciferase derived from Renilla reniformis arerepresented by SEQ ID NO: 13 and SEQ ID NO: 14. Of Oplophorusluciferase, the nucleotide sequence and amino acid sequence of theluciferase derived from Oplophorus gracilorostris are represented byrepresented by SEQ ID NO: 15 and SEQ ID NO: 16. Furthermore, of Gaussialuciferase, the nucleotide sequence and amino acid sequence of theluciferase derived from Gaussia princeps are represented by SEQ ID NO:17 and SEQ ID NO: 18.

In an embodiment of the present invention, Renilla luciferase is theluciferase derived from Renilla reniformis and comprises a polypeptideconsisting of the amino acid sequence of, e.g., SEQ ID NO: 14. Inanother embodiment of the present invention, Oplophorus luciferase isthe luciferase derived from Oplophorus gracilorostris and comprises apolypeptide consisting of the amino acid sequence of, e.g., SEQ ID NO:16. In still another embodiment of the present invention, Gaussialuciferase is the luciferase derived from Gausia princeps and comprisesa polypeptide consisting of the amino acid sequence of, e.g., SEQ ID NO:18.

When these luminescent catalysts are brought into contact withcoelenterazine analog in some embodiments of the present invention,light is produced. The emission time is generally 0.01 to 1 hour.However, the emission time can be more prolonged or the emission timecan be further shortened, depending upon conditions chosen.

(2) Detection or Quantification of Calcium Ions

The photoprotein of the present invention obtained as above is aphotoprotein (holoprotein) that non-covalent bond is formed between theapoprotein and the peroxide of coelenterazine analog produced fromcoelenterazine analog and molecular oxygen and emits light by the actionof calcium ions. Thus, the photoprotein of the invention can be used forthe detection or quantification of calcium ions.

The detection or quantification of calcium ions may be performed byadding a sample solution directly to a solution of the photoprotein andmeasuring the luminescence generated. Alternatively, calcium ions mayalso be detected or quantified by adding a solution of the photoproteinto a sample solution and measuring the luminescence generated. Thephotoprotein described above may also be previously produced, beforeaddition to the assay system for the detection or quantification ofcalcium ions, by contacting an aqueous solution of the apoprotein withcoelenterazine analog of the present invention, which is provided foruse. Alternatively, the photoprotein composed of the apoprotein and theperoxide of coelenterazine analog may be formed by contacting theapoprotein with coelenterazine analog in the assay system. Thephotoprotein formed is a complex (photoprotein) of the apoprotein andthe peroxide of coelenterazine analog of the invention. The complex(i.e., the photoprotein of the present invention) described above emitslight dependently on the calcium ion level.

The detection or quantification of calcium ions can be performed bymeasuring on a luminometer the luminescence of the photoprotein of theinvention induced by calcium ions. Luminometers which can be usedinclude commercially available instruments such as Centro LB 960(manufactured by Berthold), etc. The calcium ion level can be quantifiedby preparing a luminescence standard curve for known calcium ion levelsusing the photoprotein.

Coelenterazine analog of the present invention may also be used todetect changes in intracellular calcium ion levels under physiologicalconditions, by preparing the photoprotein composed of the apoprotein andthe peroxide of coelenterazine analog and directly introducing thephotoprotein into the cell by means of microinjection, etc.

In addition to the introduction into a cell by means of microinjection,etc., coelenterazine analog of the present invention may also be used toform the photoprotein by intracellularly expressing an apoprotein gene(a polynucleotide encoding the apoprotein) to form the apoprotein withina cell and then adding coelenterazine analog of the present invention tothe apoprotein thus formed from outside the cell.

Using the photoprotein of the invention thus introduced into or formedwithin the cell, changes in intracellular calcium ion levels in responseto external stimuli (e.g., stimuli with a drug which is associated witha receptor) can also be measured.

(3) Use as Reporter Protein, Etc. by Luminescence

The photoprotein of the present invention may also be used as a reporterprotein to determine the transcription activity of a promoter, etc. Apolynucleotide encoding the apoprotein is fused to a target promoter orother expression control sequence (e.g., an enhancer, etc.) to constructa vector. The vector is transformed to a host cell. Coelenterazine orits analog is brought into contact with the transformant. By detectingthe luminescence from the photoprotein of the present invention, theactivity of the target promoter or other expression control sequence canbe determined. As used herein, the term “contact” means that a host celland coelenterazine or its analog are allowed to be present in the sameculture system or reaction system, and includes, for example,coelenterazine or its analog being added to a culture container chargedwith a host cell, a host cell being mixed with coelenterazine or itsanalog, a host cell being cultured in the presence of coelenterazine orits analog, and the like. Coelenterazine or its analog includes thosedescribed above, in addition to coelenterazine analog of the presentinvention.

Coelenterazine analog of the present invention may be used to determinethe transcription activity of a promoter, etc. For example, apolynucleotide encoding a luminescent catalyst is fused to a targetpromoter or other expression control sequence (e.g., an enhancer, etc.)to construct a vector. The vector is transformed to a host cell.Coelenterazine analog of the present invention is brought into contactwith the transformant. By detecting the luminescence from coelenterazineanalog of the present invention, the activity of the target promoter orother expression control sequence can be determined. As used herein, theterm “contact” means that a host cell and coelenterazine analog of thepresent invention are allowed to be present in the same culture systemor reaction system, and includes, for example, coelenterazine analogbeing added to a culture container charged with a host cell, a host cellbeing mixed with coelenterazine analog, a host cell being cultured inthe presence of coelenterazine analog, and the like. The luminescentcatalyst includes those described above and is at least one selectedfrom the group consisting of Renilla luciferase, Oplophorus luciferaseand Gaussia luciferase.

The present invention further provides a kit used for measuring thetranscription activity of a promoter, etc. The kit in some embodimentsof the present invention comprises coelenterazine analog of the presentinvention and the luminescent catalyst. The kit in another embodiment ofthe present invention comprises the photoprotein of the presentinvention and coelenterazine or its analog. Reagents includingcoelenterazine or its analog, the luminescent catalyst, etc. may bedissolved in a suitable solvent and prepared into a form suitable forstorage. At least one selected from the group consisting of water,ethanol, various buffer solutions and the like may be used as thesolvent. The kit may additionally contain, if necessary, at least oneselected from the group consisting of exclusive containers, othernecessary accessories, instruction manuals, etc.

(4) Use as Detection Marker, Etc. by Luminescence

The photoprotein of the present invention can be used as a detectionmarker by luminescence. The detection marker of the present inventioncan be used to detect an analyte in, e.g., immunoassay, hybridizationassay, etc. The photoprotein of the present invention can be used in theform bound to an analyte (protein, nucleic acid, etc.) by methodsconventionally used, such as chemical modification. Detection using sucha detection marker can be carried out in a conventional manner. Thedetection marker of the invention may also be used, for example, byexpressing as a fusion protein of the apoprotein and an analyte,inserting the fusion protein into a cell by means of microinjection,etc. and contacting the protein with coelenterazine analog of theinvention to produce the photoprotein of the present invention, and thencontacting the photoprotein with coelenterazine or its analog, therebyto determine distribution of the analyte described above. As usedherein, the term “contact” means that a cell and coelenterazine analog,etc. of the present invention are allowed to be present in the sameculture system or reaction system, and includes, for example,coelenterazine analog, etc. of the present invention being added to aculture container charged with a cell, a cell being mixed withcoelenterazine analog, etc. of the present invention, a host cell beingcultured in the presence of coelenterazine analog, etc. of the presentinvention, and the like. Examples of the “coelenterazine or its analog”are those described above, in addition to coelenterazine analog of thepresent invention.

The present invention provides a method for detecting an analyte inimmunoassay, hybridization assay, etc., which comprises usingcoelenterazine analog and the luminescent catalyst. In this case, theluminescent catalyst can be used in the form bound to an analyte(protein, nucleic acid, etc.) by methods conventionally used, such aschemical modification. Detection using such a detection marker can becarried out in a conventional manner. The detection marker may also beused, for example, by expressing as a fusion protein of the luminescentcatalyst and an analyte, inserting the fusion protein into a cell bymeans of microinjection, etc. and contacting the protein withcoelenterazine analog of the invention, thus to determine distributionof the analyte described above. As used herein, the term “contact” meansthat a cell and coelenterazine analog of the present invention areallowed to be present in the same culture system or reaction system, andincludes, for example, coelenterazine analog of the present inventionbeing added to a culture container charged with a cell, a cell beingmixed with coelenterazine analog of the present invention, a host cellbeing cultured in the presence of coelenterazine analog of the presentinvention, and the like. The luminescent catalyst includes thosedescribed above and is at least one selected from the group consistingof Renilla luciferase, Oplophorus luciferase and Gaussia luciferase.

Measurement of the distribution of such an analyte may be carried outusing a detection method such as luminescence imaging, etc. Aside fromthe introduction into a cell by means of microinjection, etc., it isalso possible to use the apoprotein by expressing the same within acell.

The invention also provides a kit used for detecting an analyte in,e.g., immunoassay, hybridization assay, etc. The kit in some embodimentsof the present invention comprises the photoprotein of the invention andcoelenterazine or its analog. The kit in another embodiment of thepresent invention comprises coelenterazine analog of the invention andthe luminescent catalyst. Reagents including coelenterazine or itsanalog, the luminescent catalyst, etc. may be dissolved in a suitablesolvent and prepared into a form suitable for storage. At least oneselected from the group consisting of water, ethanol, various buffersolutions and the like may be used as the solvent. The kit mayadditionally contain, if necessary, at least one selected from the groupconsisting of exclusive containers, other necessary accessories,instruction manuals, etc.

(5) Material for Amusement Supplies

The complex (photoprotein of the present invention) composed of theapoprotein and the peroxide of coelenterazine analog of the presentinvention emits light merely by binding to a trace amount of calciumions. Therefore, the photoprotein of the invention can be advantageouslyused as a luminescence substrate in materials for amusement supplies.Examples of amusement supplies include luminescent bubble soap,luminescent ice, luminescent candies, luminescent paints, etc. Theamusement supplies of the invention can be prepared in a conventionalmanner.

(6) Bioluminescence Resonance Energy Transfer (BRET) Method

Coelenterazine analog in some embodiments of the present invention emitslight by the action of the luminescent catalyst as described above andcan thus be used for the method of analyses, including an analysis ofbiological functions, an analysis (or measurement) of enzyme activities,etc., based on the principle of intermolecular interactions by thebioluminescence resonance energy transfer (BRET) method. In addition,the photoprotein of the present invention can also be used for themethod of analyses such as an analysis of biological functions,measurement of enzyme activities, etc., based on the principle ofintermolecular interactions by the bioluminescence resonance energytransfer (BRET) method.

For example, using coelenterazine analog of the present invention insome embodiments of the invention and the luminescent catalyst as donorproteins and an organic compound or a fluorescent protein as anacceptor, the interactions between the proteins can be detected bycausing bioluminescence resonance energy transfer (BRET) between them.Herein, the luminescent catalyst includes those described above and isat least one selected from the group consisting of Renilla luciferase,Oplophorus luciferase and Gaussia luciferase. Alternatively, using thephotoprotein of the invention as a donor protein and an organic compoundor fluorescent protein an acceptor, the interactions between theproteins can be detected by causing bioluminescence resonance energytransfer (BRET) between them. In an embodiment of the present invention,the organic compound used as an acceptor is Hoechst 3342, Indo-1, DAP1,etc. In another embodiment of the present invention, the fluorescentprotein used as an acceptor is a green fluorescent protein (GFP), a bluefluorescent protein (BFP), a mutant GFP fluorescent protein, phycobilin,etc. In a preferred embodiment of the present invention, thephysiological functions to be analyzed include an orphan receptor (inparticular, a (i-protein coupled receptor), apoptosis, transcriptionregulation by gene expression, etc. In a preferred embodiment of thepresent invention, the enzyme to be analyzed is protease, esterase,kinase, or the like.

Analysis of the physiological functions by the BRET method may beperformed by publicly known methods, for example, by modifications ofthe methods described in Biochem. J. 2005, 385, 625-637, Expert Opin.Ther Tarets, 2007 11: 541-556, etc. Assay for the enzyme activity mayalso be performed by publicly known methods, for example, bymodifications of the methods described in Nat Methods 2006, 3:165-174,Biotechnol J. 2008, 3:311-324, etc.

Furthermore, the present invention provides a kit used for the method ofanalysis described above. The kit comprises coelenterazine analog of thepresent invention, the luminescent catalyst, and the organic compoundand/or fluorescent protein. Alternatively, the kit comprises thephotoprotein of the present invention and the organic compound and/orfluorescent protein. Reagents including coelenterazine analog, theluminescent catalyst, the photoprotein of the invention, the organiccompound, the fluorescent protein, etc. may be dissolved in a suitablesolvent and prepared into a form suitable for storage. At least oneselected from the group consisting of water, ethanol, various buffersolutions and the like may be used as the solvent. The kit mayadditionally contain, if necessary, at least one selected from the groupconsisting of exclusive containers, other necessary accessories,instruction manuals, etc.

All literatures and publications mentioned in this specification areherein incorporated in their entirety by reference into thespecification, irrespective of their purposes. The specificationincludes all of the contents as disclosed in the claims, specificationand drawings of Japanese Patent Application No. 2009-27921 (filed Feb.9, 2009), based on which the priority of the present application isenjoyed.

The objects, characteristics, and advantages of the present invention aswell as the idea thereof are apparent to those skilled in the art fromthe descriptions given herein, and those skilled in the art can easilyimplement the present invention. It is to be understood that the bestmode to carry out the invention and specific examples are to be taken aspreferred embodiments of the present invention. These descriptions areonly for illustrative and explanatory purposes and are not intended torestrict the invention thereto. It is further apparent to those skilledin the art that various modifications may be made based on thedescriptions given herein within the intent and scope of the presentinvention disclosed herein.

In the following EXAMPLES, the ratios of solvent mixtures forchromatography are all by v/v unless otherwise indicated.

EXAMPLES Synthesis Examples Outline of Synthesis of CoelenterazineAnalog (CTZ Analog)

The outline of synthesis for i-coelenterazine (i-CTZ (R¹=1, R²=H,R³=H)), n-coelenterazine (n-CTZ (R¹, R²=benzo, R³=H)), me-coelenterazine(me-CTZ (R¹=CH₃, R²=H, R³=H)), et-coelenterazine (et-CTZ (R¹=C₂H₅, R²=H,R³=H)), cf3-coelenterazine (cf3-CTZ (R¹=CF₃, R²=H, R³=H),meo-coelenterazine (meo-CTZ (R¹=OCH₃, R²=H, R³=H)), 3me-coelenterazine(3me-CTZ (R¹=H, R²=CH₃, R³=H)), 3meo-coelenterazine (3meo-CTZ (R¹=H,R²=OCH₃, R³=H)), αmeh-coelenterazine (αmeh-CTZ (R¹=H, R²=H, R³=CH₃)) and3-isocoelenterazine (3iso-CTZ (R¹=H, R²=OH, R³=H)) using a keto acetalis as shown below.

The outline of synthesis fir i-coelenterazine (i-CTZ) using aketoaldehyde is as shown below.

Process

In the synthesis of CTZ analogs, flash column chromatography wasperformed using silica gel (37563-85 manufactured by Kanto Chemical Co.,Inc., Silica Gel 60N (spherical, neutral), and particle size of 40-50μm), except that acidic silica gel (37562-79 manufactured by KantoChemical Co., Inc., Silica Gel 60 (spherical), and particle size of40-50 μm) was used for the purification of CTZ analogs.

Thin layer chromatography (TLC) was performed using a glass plate(1.05715 manufactured by MERCK Inc., Silica Gel 60 F₂₅₄) precoated withsilica gel.

The purity of CTZ analogs was confirmed using high performance liquidchromatography (HPLC): 1100 Series HPLC System manufactured by AgilentInc.: measurement conditions, column: Lichrosorb (registered trademark)RP-18 (5 μm, 4.0 mm i.d.×125 mm, manufactured by Merck Chemicals);moving phase: gradient 60-100% methanol/0.1% aqueous TFA for 40 min;flow rate: 0.45 mL/min; detection: UV 225 nm; volume of injectionsample: 0.5 mg/5 mL in methanol/0.1% aqueous TFA=6/4.

Melting point (Mp) was measured on a micro melting point determinationapparatus MP-J3 manufactured by YANACO, Inc. (uncorrected data)

Ultraviolet absorption spectra (UV) of CTZ analogs (20 μM methanolsolution) were measured at 25° C. in quartz cells (optical path lengthof 10 mm) with UV-3100 UV-Visible-Near-Infrared Spectrophotometermanufactured by SHIMADZU Corporation under the conditions of high speedat scan speed.

Fluorescence spectra (FL) of CTZ analogs (8 μg/mL methanol) weremeasured at 25° C. in quartz cells (optical path length of 10 mm) withFP-6500 spectrofluorimeter manufactured by JASCO, under the conditionsfor excitation wavelength of 330 nm, excitation side bandwidth of 3 nm,fluorescence emission side of 3 nm, response of 0.5 second, sensitivityof medium and scan speed of 100 nm/min.

¹H NMR spectra were obtained with a Unity Plus 400 nuclear magneticresonance spectrometer manufactured by Varian Corp. ¹³C NMR spectra wereobtained with a JNM-EX270 nuclear magnetic resonance spectrometermanufactured by JEOL Co., Ltd. ¹⁹F NMR spectra were obtained with aMercury 300 spectrometer manufactured by Varian Corp. CDCl₃ or CD₃OD(both manufactured by CIL Inc.) was used as a solvent for themeasurement of NMR spectra.

Chemical shifts (δ) were expressed in terms of relative values using, asan internal standard, tetramethylsilane ((CH₃)₄Si) (measurements of ¹HNMR in CDCl₃; δ 0 ppm), the peak derived from non-deuterated solvent formeasurements (measurements of ¹H NMR in CD₃OD; δ 3.31 ppm, measurementsof ¹³C NMR in CDCl₃; δ 77.0 ppm) or hexafluorobenzene (measurements of¹⁹F NMR; δ 0 ppm). The binding constant (J) was shown by Hz.Abbreviations s, d, t, q, m and br denote singlet, doublet, triplet,quartet, multiplet, and broad, respectively.

Infrared spectroscopic spectra (IR) were measured by diffuse reflectancemethod on an IRPrestige-21 Fourier Transform Infrared Spectrophotometermanufactured by SHIMADZU Corporation, equipped with a DRS-8000A diffusereflectance measuring device.

High resolution mass spectrometric spectra (HRMS) were measured onJMS-700 manufactured by JEOL by the electron impact ionization (EI⁺)method, or the fast atom bombardment (FAB⁺) method. In the FAB⁺ method,m-nitrobenzyl alcohol (NBA) or glycerol was used as a matrix.

Synthesis Example 1 Synthesis of i-coelenterazine (i-CTZ)

Synthesis Example 1-1

Under an argon atmosphere, to 4-iodophenylacetic acid (11) (prepared bythe process described in Chen, Q.-H. et al., Bioorg. Med. Chem. 14,7898-7909 (2006)) (1.06 g, 4.05 mmol) was added thionyl chloride (5.00mL, 68.6 mmol) and heated to reflux (100° C.) for 1.5 h. After coolingto room temperature, the mixture was concentrated under reduced pressureto give 4-iodophenylacetyl chloride (12) as a brown oily crude product,which was used in the next reaction without further purification.

Synthesis Example 1-2

Under an argon atmosphere, 4-iodophenylacetyl chloride (12) preparedabove was dissolved in tetrahydrofuran (THF) (2 mL) and acetonitrile (2mL) and cooled to 0° C. To this was slowly added a solution oftrimethylsilyldiazomethane in diethyl ether (2.0 M, 4.00 mL, 8.00 mmol),which was stirred overnight (14 h) after warming up to room temperature.After concentrating under reduced pressure, the residue was purified bysilica gel flash column chromatography (n-hexane/diethyl ether=1/1) togive 1-diazo-3-(4-iodophenyl)propan-2-one (13) as a pale yellow solid(635 mg, 2.22 mmol, 54.9%, 2 steps).

TLC R_(f)=0.34 (n-hexane/diethyl ether=1/2);

¹H NMR (400 MHz, CDCl₃) δ 3.55 (s, 2H), 5.14 (s, 1H), 6.97-7.01 (AA′BB′,2H), 7.65-7.69 (AA′BB′, 2H);

¹³C NMR (67.8 MHz, CDCl₃) δ 47.3, 55.1, 92.9, 131.4 (2C), 134.2, 137.9(2C), 191.9;

IR (KBr, cm⁻¹) 737, 802, 841, 1007, 1138, 1306, 1371, 1402, 1483, 1630,2102, 2114, 3076;

HRMS (EI) m/z 285.9597 (M, C₉H₇ON₂O required 285.9603).

Synthesis Example 1-3

Under an argon atmosphere, 1-diazo-3-(4-iodophenyl)propan-2-one (13)(1.11 g, 3.88 mmol) was dissolved in anhydrous ethanol (10 mL) andcooled to 0° C. To this was added tert-butyl hypochlorite (440 μL, 3.89mmol) and stirred for an hour at the same temperature. Afterconcentrating under reduced pressure, the residue was purified by silicagel flash column chromatography (n-hexane/ethyl acetate=9/1) to give1,1-diethoxy-3-(4-iodophenyl)propan-2-one (14) as a yellow oilysubstance (758 mg, 2.18 mmol, 56.1%).

TLC R_(f)=0.27 (n-hexane/ethyl acetate=9/1);

¹H NMR (400 MHz, CDCl₃) δ 1.25 (t, 6H, J=7.0 Hz), 3.55 (dq, 2H, J=9.5,7.0 Hz), 3.71 (dq, 2H, J=9.5, 7.0 Hz), 3.83 (s, 2H), 4.61 (s, 1H),6.94-6.98 (AA′BB′, 2H), 7.62-7.66 (AA′BB′, 2H);

¹³C NMR (67.8 MHz, CDCl₃) δ 15.2 (2C), 43.0, 63.6 (2C), 92.5, 102.6,131.9 (2C), 133.5, 137.6 (2C), 202.7;

IR (KBr, cm⁻¹) 718, 1007, 1061, 1098, 1157, 1315, 1369, 1400, 1443,1485, 1584, 1643, 1732, 2882, 2928, 2976, 3321;

HRMS (FAB+/NBA) m/z 349.0303 (M+H, C₁₃H₁₈IO₃ required 349.0301).

Synthesis Example 1-4

Under an argon atmosphere, 1,1-diethoxy-3-(4-iodophenyl)propan-2-one(14) (421 mg, 1.21 mmol) was dissolved in 1,4-dioxane (2 mL) and water(0.4 mL). To this was added coelenteramine (prepared by the processdescribed in Adamczyk, M. et al., Org. Prep. Proced. Int., 33, 477-485(2001)) (222 mg, 801 μmol) and, after cooling to 0° C., conc.hydrochloric acid (0.20 mL) was further added thereto, and then stirredovernight (15 h) at 100° C. After cooling to room temperature, themixture was concentrated under reduced pressure and the residue waspurified by silica gel flash chromatography in an argon flow(n-hexane/ethyl acetate=1/2→ethyl acetate→ethylacetate/methanol=20/1→10/1). The solid obtained was furtherreprecipitated (n-hexane/acetone) to give i-coelenterazine (15, i-CTZ)as an ocher powder (45.2 mg, 84.7 μmol, 10.6%).

TLC R_(f)=0.52 (ethyl acetate/methanol=20/1);

HPLC retention time 21.5 min;

Mp 157-159° C. (dec.);

UV (MeOH) λmax (ε)=259 (24900), 343 (5500), 432 (8300) nm;

FL (MeOH) λmax Em. 545.5 nm;

¹H NMR (400 MHz, CD₃OD) δ 4.18 (s, 2H), 4.48 (s, 2H), 6.88-6.92 (AA′BB′,2H), 7.09-7.13 (AA′BB′, 2H), 7.22-7.42 (m, 5H), 7.55-7.60 (AA′BB′, 2H),7.60-7.66 (AA′BB′, 2H), 7.94 (br, ¹H);

IR (KBr, cm⁻¹) 700, 839, 1007, 1171, 1236, 1277, 1506, 1541, 1558, 1609,1647, 2810, 2934, 2965, 3030, 3059;

HRMS (EI) m/z 533.0587 (M, C₂₆H₂₀IN₃O₂ required 533.0600).

Synthesis Example 2 Synthesis of n-coelenterazine (n-CTZ)

Synthesis Example 2-1

Under an argon atmosphere, to a solution of ethyl diethoxyacetate (900μL, 5.06 mmol) in THF (15 mL) was added slowly a diethyl ether solutionof (2-naphtalenylmethyl)magnesium bromide (21) (0.25 M, 25.0 mL, 6.25mmol) at −78° C. After stirring at for 2.5 h at −78° C., the mixture wasgradually warmed to room temperature and stirred for 15 h. To this wasadded 20% aqueous solution of ammonium chloride (10 mL) and the productwas extracted with ethyl acetate (×3). The organic layer wassequentially washed with water (×1) and saturated brine (×1), and driedover anhydrous sodium sulfate. After filtration, the mixture wasconcentrated under reduced pressure and the residue was purified bysilica gel flash column chromatography (n-hexane/ethyl acetate=19/1) togive 1,1-diethoxy-3-(2-naphthalenyl)propan-2-one (22) as a colorlessoily substance (675 mg, 2.48 mmol, 49.0%).

TLC R_(f)=0.37 (n-hexane/ethyl acetate=9/1);

¹H NMR (400 MHz, CDCl₃) δ 1.26 (t, 6H, J=7.0 Hz), 3.56 (dq, 2H, J=9.5,7.0 Hz), 3.72 (dq, 2H, J=9.5, 7.0 Hz), 4.06 (s, 2H), 4.66 (s, 1H), 7.35(dd, 1H, J=1.8, 8.5 Hz), 7.42-7.49 (m, 2H), 7.69 (s, 1H), 7.76-7.84 (m,3H);

¹³C NMR (δ 7.8 MHz, CDCl₃) δ 15.2 (2C), 43.9, 63.5 (2C), 102.4, 125.7,126.1, 127.66, 127.69, 128.0, 128.1, 128.5, 131.4, 132.4, 133.5, 203.2;

IR (KBr, cm⁻¹) 812, 1017, 1061, 1098, 1125, 1310, 1370, 1508, 1732,2882, 2928, 2976, 3053;

HRMS (FAB⁺/NBA+NaI) m/z 295.1316 (M+Na, C₁₇H₂₀O₃Na required 295.1310).

Synthesis Example 2-2

Under an argon atmosphere, 1,1-diethoxy-3-(2-naphthalenyl)propan-2-one(22) (369 mg, 1.35 mmol) was dissolved in 1,4-dioxane (2 mL) and water(0.4 mL). To this was added coelenteramine (310 mg, 1.12 mmol) and,after cooling to 0° C., conc. hydrochloric acid (0.20 mL) was furtheradded thereto, and then stirred at 100° C. overnight (15 hours). Aftercooling to room temperature, the mixture was concentrated under reducedpressure and the residue was purified by silica gel flash chromatographyin an argon flow (n-hexane/ethyl acetate=1/2→ethyl acetate→ethylacetate/methanol=50/1→20/1). The solid obtained was furtherreprecipitated (n-hexane/acetone) to give n-coelenterazine (23, n-CTZ)as a yellow powder (88.5 mg, 193 μmol, 17.3%).

TLC R_(f)=0.47 (ethyl acetate/methanol=20/1);

HPLC retention time 20.6 min;

Mp 149-151° C. (dec.);

UV (MeOH) λ_(max) (ε)=262.5 (34500), 349.5 (6400), 432.5 (11700) nm;

FL (MeOH) λ_(max) Em. 434.5, 544 nm;

¹H NMR (400 MHz, CD₃OD) δ 4.35 (s, 2H), 4.41 (s, 2H), 6.85-6.91 (AA′BB′,2H), 7.18-7.31 (m, 3H), 7.35-7.45 (m, 4H), 7.46-7.54 (m, 3H), 7.68-7.80(m, 5H);

IR (KBr, cm⁻¹) 698, 760, 815, 839, 1173, 1242, 1277, 1456, 1508, 1541,1558, 1611, 2812, 2967, 3055, 3152;

HRMS (EI) m/z 457.1778 (M, C₃₀H₂₃N₃O₂ required 457.1790).

Synthesis Example 3 Synthesis of me-coelenterazine (me-CTZ)

Synthesis Example 3-1

The magnesium turnings (270 mg, 11.1 mmol) were dried in vacuo byheating with a heat gun. After cooling to room temperature and beingplaced under an argon atmosphere, THF (8 mL) was added thereto, followedby slow addition of 4-methylbenzyl chloride (31) (1.35 mL, 10.2 mmol) atroom temperature. The reaction mixture became warm as the result of anexothermic reaction and most of the magnesium turnings were consumed.After cooling to room temperature, it was used directly in the nextreaction as a THF solution of (4-methylbenzyl)magnesium chloride (32).

Under an argon atmosphere, to a solution of ethyl diethoxyacetate (1.80mL, 10.1 mmol) in THF (20 mL) was added slowly a THF solution of (32)prepared above at −78° C. After stirring at −78° C. for an hour, to thiswas added 20% aqueous solution of ammonium chloride (10 mL) and theproduct was extracted with ethyl acetate (×3). The organic layer wassequentially washed with water (×1) and saturated brine (×1), and driedover anhydrous sodium sulfate. After filtration, the mixture wasconcentrated under reduced pressure and the residue was purified bysilica gel flash column chromatography (n-hexane/ethyl acetate=9/1) togive 1,1-diethoxy-3-(4-methylphenyl)propan-2-one (33) as a colorlessoily substance (1.96 g, 8.27 mmol, 82.4%).

TLC R_(f)=0.45 (n-hexane/ethyl acetate=9/1);

¹H NMR (400 MHz, CDCl₃) δ 1.25 (t, 6H, J=7.0 Hz), 2.32 (s, 3H), 3.55(dq, 2H, J=9.5, 7.0 Hz), 3.70 (dq, 2H, J=9.5, 7.0 Hz), 3.85 (s, 2H),4.63 (s, 1H), 7.08-7.14 (2AA′BB′, 4H);

¹³C NMR (67.8 MHz, CDCl₃) δ 15.1 (2C), 21.0, 43.3, 63.3 (2C), 102.2,129.1 (2C), 129.6 (2C), 130.6, 136.3, 203.3;

IR (KBr, cm⁻¹) 772, 804, 851, 1022, 1063, 1098, 1146, 1312, 1516, 1732,2884, 2926, 2976;

HRMS (EI) m/z 236.1409 (M, C₁₄H₂₀O₃ required 236.1412).

Synthesis Example 3-2

Under an argon atmosphere, 1,1-diethoxy-3-(4-methylphenyl)propan-2-one(33) (216 mg, 914 μmol) was dissolved in 1,4-dioxane (2 mL) and water(0.4 mL). To this was added coelenteramine (196 mg, 707 μmol) and, aftercooling to 0° C., conc. hydrochloric acid (0.20 mL) was further addedthereto, and then stirred overnight (14 hours) at 100° C. After coolingto room temperature, the mixture was concentrated under reduced pressureand the residue was purified by silica gel flash chromatography in anargon flow (n-hexane/ethyl acetate=1/2→ethyl acetate→ethylacetate/methanol=50/1→20/1). The solid obtained was furtherreprecipitated (n-hexane/acetone) to give me-coelenterazine (34, me-CTZ)as a yellow powder (180 mg, 427 μmol, 60.4%).

TLC R_(f)=0.51 (ethyl acetate/methanol=20/1);

HPLC retention time 17.1 min;

Mp 150-152° C. (dec.);

UV (MeOH) λ_(max) (ε)=260 (20900), 351 (4600), 437.5 (8600) nm;

FL (MeOH) λ_(max) Em. 433.5, 545 nm;

¹H NMR (400 MHz, CD₃OD) δ2.29 (s, 3H), 4.22 (s, 2H), 4.52 (s, 2H),6.88-6.93 (AA′BB′, 2H), 7.09-7.14 (AA′BB′, 2H), 7.15-7.20 (AA′BB′, 2H),7.22-7.34 (m, 3H), 7.38-7.42 (m, 2H), 7.67-7.73 (AA′BB′, 2H), 8.23 (br,¹H);

IR (KBr, cm⁻¹) 700, 820, 843, 1171, 1238, 1279, 1508, 1541, 1589, 1609,2812, 2924, 3028;

HRMS (FAB⁺/glycerol) m/z 422.1880 (M+H, C₂₇H₂₄N₃O₂ required 422.1869).

Synthesis Example 4 Synthesis of et-coelenterazine (et-CTZ) SynthesisExample 4-1

The magnesium turnings (273 mg, 11.2 mmol) were dried in vacuo byheating with a heat gun. After cooling to room temperature and beingplaced under an argon atmosphere, THF (8 mL) was added thereto, followedby slow addition of 4-ethylbenzyl chloride (41) (1.48 mL, 9.95 mmol) atroom temperature. The reaction mixture became warm as the result of anexothermic reaction and most of magnesium turnings were consumed. Aftercooling to room temperature, it was used directly in the next reactionsas a THF solution of (4-ethylbenzyl)magnesium chloride (42).

Under an argon atmosphere, to a solution of ethyl diethoxyacetate (1.80mL, 10.1 mmol) in THF (20 mL) was added slowly a THF solution (42) of(4-ethylbenzyl)magnesium chloride prepared above at −78° C. Afterstirring at −78° C. for an hour, to this was added 20% aqueous solutionof ammonium chloride (10 mL), and the product was extracted with ethylacetate (×3). The organic layer was sequentially washed with water (×1)and saturated brine (×1), and dried over anhydrous sodium sulfate. Afterfiltration, the mixture was concentrated under reduced pressure and theresidue was purified by silica gel flash column chromatography(n-hexane/ethyl acetate=9/1) to give1,1-diethoxy-3-(4-ethylphenyl)propan-2-one (43) as a colorless oilysubstance (1.29 g, 5.04 mmol, 50.6%).

TLC R_(f)=0.43 (n-hexane/ethyl acetate=9/1)

¹H NMR (400 MHz, CDCl₃)□ δ 1.22 (t, 3H, J=7.6 Hz), 1.25 (t, 6H, J=7.0Hz), 2.63 (q, 2H, J=7.6 Hz), 3.55 (dq, 2H, J=9.5, 7.0 Hz), 3.70 (dq, 2H,J=9.5, 7.0 Hz), 3.86 (s, 2H), 4.63 (s, 1H), 7.10-7.17 (2AA′BB′, 4H);

¹³C NMR (67.8 MHz, CDCl₃) δ 15.1 (2C), 15.5, 28.4, 43.2, 63.2 (2C),102.2, 127.9 (2C), 129.6 (2C), 130.8, 142.6, 203.2;

IR (KBr, cm⁻¹) 810, 853, 1022, 1061, 1099, 1151, 1314, 1514, 1732, 2874,2930, 2974;

HRMS (EI) m/z 250.1566 (M, C₁₅H₂₂O₃ required 250.1569).

Synthesis Example 4-2

Under an argon atmosphere, 1,1-diethoxy-3-(4-ethylphenyl)propan-2-one(43) (301 mg, 1.20 mmol) was dissolved in 1,4-dioxane (2 mL) and water(0.4 mL). To this was added coelenteramine (238 mg, 858 μmol) and, aftercooling to 0° C., conc. hydrochloric acid (0.20 mL) was further addedthereto, and then stirred overnight (17 hours) at 100° C. After coolingto room temperature, the mixture was concentrated under reduced pressureand the residue was purified by silica gel flash chromatography in anargon flow (n-hexane/ethyl acetate=1/2→ethyl acetate→ethylacetate/methanol=50/1→10/1). The solid obtained was furtherreprecipitated (n-hexane/acetone) to give et-coelenterazine (44, et-CTZ)as a yellow powder (240 mg, 551 μmol, 64.2%).

TLC R_(f)=0.52 (ethyl acetate/methanol=20/1);

HPLC retention time 20.5 min;

Mp 145-147° C. (dec.);

UV (MeOH) λ_(max) (ε)=259.5 (22300), 342.5 (5000), 434.5 (8800) nm;

FL (MeOH) λ_(max) Em. 433, 546.5 nm;

¹H NMR (400 MHz, CD₃OD) δ 1.19 (t, 3H, J=7.6 Hz), 2.60 (q, 2H, J=7.6Hz), 4.24 (s, 2H), 4.53 (s, 2H), 6.88-6.96 (AA′BB′, 2H), 7.17-7.22(2AA′BB′, 4H), 7.24-7.34 (m, 3H), 7.38-7.43 (m, 2H), 7.69-7.75 (AA′BB′,2H), 8.26 (br, ¹H);

IR (KBr, cm⁻¹) 700, 820, 840, 1171, 1238, 1277, 1508, 1541, 1589, 1609,1647, 2893, 2930, 2963, 3028;

HRMS (FAB⁺/glycerol) m/z 436.2026 (M+H, C₂₈H₂₆N₃O₂ required 436.2025).

Synthesis Example 5 Synthesis of cf3-coelenterazine (cf3-CTZ)

Synthesis Example 5-1

Under an argon atmosphere, to 4-trifluoromethylphenylacetic acid (51)(817 mg, 4.00 mmol) was added thionyl chloride (5.00 mL, 68.9 mmol), andheated to reflux (100° C.) for 1.5 h. After cooling to room temperature,the mixture was concentrated under reduced pressure to give4-trifluoromethylphenylacetyl chloride (52) as a brown oily crudeproduct, which was used in the next reaction without furtherpurification.

Synthesis Example 5-2

Under an argon atmosphere, p-trifluoromethylphenylacetyl chloride (52)prepared above was dissolved in THE (2 mL) and acetonitrile (2 mL), andcooled to 0° C. To this was slowly added a solution oftrimethylsilyldiazomethane in diethyl ether (2.0 M, 4.00 mL, 8.00 mmol),which was stirred overnight (20 hours) after warming up to roomtemperature. After being concentrated under reduced pressure, theresidue was purified by silica gel flash column chromatography(n-hexane/diethyl ether=1/1) to give1-diazo-3-[4-(trifluoromethyl)phenyl]propan-2-one (53) as a pale yellowoily substance (666 mg, 2.92 mmol, 72.9%, 2 steps).

TLC R_(f)=0.31 (n-hexane/diethyl ether=1/2);

¹H NMR (400 MHz, CDCl₃) δ 3.68 (s, 2H), 5.18 (s, 1H), 7.34-7.40 (AA′BB′,2H), 7.59-7.63 (AA′BB′, 2H);

¹³C NMR (67.8 MHz, CDCl₃) δ 47.4, 55.3, 124.2 (q, ¹J_(C—F)=271.9 Hz),125.7 (2C, q, ³J_(C—F)=3.8 Hz), 129.1 (q, ²J_(C—F)=32.4 Hz), 129.8 (2C),138.6, 191.4;

¹⁹F NMR (282 MHz, CDCl₃) δ 99.3 (s);

IR (KBr, cm⁻¹) 743, 824, 854, 1020, 1067, 1119, 1164, 1325, 1366, 1420,1620, 1634, 1639, 2107, 3086;

HRMS (EI) m/z 228.0511 (M, C₁₀H₇F₃N₂O required 228.0510).

Synthesis Example 5-3

Under an argon atmosphere,1-diazo-3-[4-(trifluoromethyl)phenyl]propan-2-one (53) (661 mg, 2.90mmol) was dissolved in anhydrous ethanol (6 mL) and cooled to 0° C. Tothis was added tert-butyl hypochlorite (330 μL, 2.92 mmol) and stirredfor an hour at the same temperature. After being concentrated underreduced pressure, the residue was purified by silica gel flash columnchromatography (n-hexane/ethyl acetate=18/1) to give1,1-diethoxy-3-[4-(trifluoromethyl)phenyl]propan-2-one (54) as acolorless oily substance (544 mg, 1.87 mmol, 64.7%).

TLC R_(f)=0.29 (n-hexane/ethyl acetate=9/1);

¹H NMR (400 MHz, CDCl₃) δ 1.26 (t, 6H, J=7.0 Hz), 3.57 (dq, 2H, J=9.2,7.0 Hz), 3.74 (dq, 2H, J=9.2, 7.0 Hz), 3.96 (s, 2H), 4.62 (s, 1H),7.30-7.35 (AA′BB′, 2H), 7.54-7.60 (AA′BB′, 2H);

¹³C NMR (67.8 MHz, CDCl₃) δ 15.2 (2C), 43.0, 63.7 (2C), 102.8, 124.3 (q,¹J_(C—F)=271.9 Hz), 125.3 (2C, q, ³J_(C—F)=3.8 Hz), 129.6 (q,²J_(C—F)=32.4 Hz), 130.3 (2C), 138.1, 202.4;

¹⁹F NMR (282 MHz, CDCl₃) δ 99.3 (s);

IR (KBr, cm⁻¹) 864, 1020, 1067, 1109, 1125, 1165, 1325, 1732, 2884,2934, 2980;

HRMS (FAB⁺/NBA+NaI) m/z 313.1031 (M+Na, C₁₄H₁₇F₃O₃Na required 313.1027).

Synthesis Example 5-4

Under an argon atmosphere,1,1-diethoxy-3-[4-(trifluoromethyl)phenyl]propan-2-one (54) (359 mg,1.24 mmol) was dissolved in 1,4-dioxane (2 mL) and water (0.4 mL). Tothis was added coelenteramine (217 mg, 782 μmol) and, after cooling to0° C., conc. hydrochloric acid (0.20 mL) was further added thereto, andthen stirred overnight (17 hours) at 100° C. After cooling to roomtemperature, the mixture was concentrated under reduced pressure and theresidue was purified by silica gel flash chromatography in an argon flow(n-hexane/ethyl acetate=1/2→ethyl acetate→ethylacetate/methanol=20/1→10/1). The solid obtained was furtherreprecipitated (n-hexane/acetone) to give cf3-coelenterazine (55,cf3-CTZ) as a yellow powder (232 mg, 488 μmol, 62.4%).

TLC R_(f)=0.30 (ethyl acetate/methanol=20/1);

HPLC retention time 19.9 min;

Mp 157-161° C. (dec.);

UV (MeOH) λ_(max) (ε)=259 (27000), 341.5 (5600), 440.5 (10500) nm;

FL (MeOH) λ_(max) 549.5 nm;

¹H NMR (400 MHz, CD₃OD) δ 4.37 (s, 2H), 4.55 (s, 2H), 6.90-6.95 (AA′BB′,2H), 7.23-7.34 (m, 3H), 7.39-7.43 (m, 2H), 7.49-7.53 (AA′BB′, 2H),7.61-7.71 (2AA′BB′, 4H), 8.23 (br, ¹H);

¹⁹F NMR (282 MHz, CDCl₃) δ 101.8 (s);

IR (KBr, cm⁻¹) 702, 818, 1018, 1067, 1121, 1177, 1327, 1508, 1541, 1593,1609, 1655, 2814, 2862, 2928, 3032, 3256;

HRMS (FAB⁺/glycerol) m/z 476.1580 (M+H, C₂₇H₂₁F₃N₃O₂ required 476.1586).

Synthesis Example 6 Synthesis of meo-coelenterazine (meo-CTZ)

Synthesis Example 6-1

Under an argon atmosphere, 4-methoxyphenylacetyl chloride (61) (952 mg,5.16 mmol) was dissolved in THF (2.5 mL) and acetonitrile (2.5 mL) andcooled to ° C. To this was slowly added a solution oftrimethylsilyldiazomethane in diethyl ether (2.0 M, 5.00 mL, 10.0 mmol),which was stirred overnight (15 hours) after warming up to roomtemperature. After concentrating under reduced pressure, the residue waspurified by silica gel flash column chromatography (n-hexane/diethylether=1/1) to give 1-diazo-3-(4-methoxyphenyl)propan-2-one (62) as apale yellow oily substance (692 mg, 3.64 mmol, 70.6%).

TLC R_(f)=0.41 (n-hexane/diethyl ether=1/2);

¹H NMR (400 MHz, CDCl₃) δ 3.56 (s, 2H), 3.81 (s, 3H), 5.11 (s, 1H),6.85-6.90 (AA′BB′, 2H), 7.12-7.18 (AA′BB′, 2H);

¹³C NMR (67.8 MHz, CDCl₃) δ 47.3, 54.7, 55.3, 114.3 (2C), 126.6, 130.5(2C), 158.9, 193.4;

IR (KBr, cm⁻¹) 821, 851, 943, 1032, 1117, 1179, 1248, 1358, 1512, 1611,1632, 2102, 2835, 2907, 2934, 3098, 3530;

HRMS (EI) m/z 190.0743 (M, C₁₀H₁₀N₂O₂ required 190.0742).

Synthesis Example 6-2

Under an argon atmosphere, 1-diazo-3-(4-methoxyphenyl)propan-2-one (62)(477 mg, 2.51 mmol) was dissolved in anhydrous ethanol (5 mL) and cooledto 0° C. To this was added tert-butyl hypochloride (285 μL, 2.52 mmol)and stirred for an hour at the same temperature. After concentratingunder reduced pressure, the residue was purified by silica gel flashcolumn chromatography (n-hexane/ethyl acetate=10/1) to give1,1-diethoxy-3-(4-methoxyphenyl)propan-2-one (63) as a colorless oilysubstance (357 mg, 1.41 mmol, 56.4%).

TLC R=0.29 (n-hexane/ethyl acetate=9/1);

¹H NMR (400 MHz, CDCl₃) δ 1.25 (t, 6H, J=7.0 Hz), 3.55 (dq, 2H, J=9.5,7.0 Hz), 3.70 (dq, 2H, J=9.5, 7.0 Hz), 3.79 (s, 3H), 3.83 (s, 2H), 4.63(s, 1H), 6.83-6.91 (AA′BB′, 2H), 7.10-7.17 (AA′BB′, 2H);

¹³C NMR (67.8 MHz, CDCl₃) δ 15.2 (2C), 42.9, 55.3, 63.4 (2C), 102.3,114.0 (2C), 125.8, 130.8 (2C), 158.6, 203.6;

IR (KBr, cm⁻¹) 1036, 1063, 1098, 1177, 1512, 1612, 1732, 2835, 2897,2933, 2976;

HRMS (EI) m/z 252.1360 (M, C₁₄H₂₀O₄ required 252.1362).

Synthesis Example 6-3

Under an argon atmosphere, 1,1-diethoxy-3-(4-methoxyphenyl)propan-2-one(63) (355 mg, 1.41 mmol) was dissolved in 1,4-dioxane (2 mL) and water(0.4 mL). To this was added coelenteramine (221 mg, 797 μmol) and, aftercooling to 0° C., conc. hydrochloric acid (0.20 mL) was further addedthereto, and then stirred overnight (17 hours) at 100° C. After coolingto room temperature, the mixture was concentrated under reduced pressureand the residue was purified by silica gel flash chromatography in anargon flow (n-hexane/ethyl acetate=1/2→ethyl acetate→ethylacetate/methanol=20/1→10/1). The solid obtained was furtherreprecipitated (n-hexane/acetone) to give meo-coelenterazine (64,meo-CTZ) as an ocher powder (174 mg, 398 μmol, 49.9%).

TLC R_(f)=0.31 (ethyl acetate/methanol==20/1);

HPLC retention time 13.7 min;

Mp 137-139° C. (dec.);

UV (MeOH) λ_(max) (ε)=267 (26000), 344.5 (7400), 435 (8500) nm;

FL (MeOH) λ_(max) Em. 435.5, 549 nm;

¹H NMR (400 MHz, CD₃OD) δ 3.75 (s, 3H), 4.21 (s, 2H), 4.54 (s, 2H),6.84-6.94 (2AA′BB′, 4H), 7.18-7.36 (m, 5H), 7.38-7.43 (m, 2H), 7.69-7.75(AA′BB′, 2H), 8.31 (br, ¹H);

IR (KBr, cm⁻¹) 820, 839, 1177, 1248, 1508, 1558, 1585, 1609, 1647, 2835,2951, 3030, 3063;

HRMS (FAB⁺/glycerol) m/z 438.1814 (M+H, C₂₇H₂₄N₃O₃ required 438.1818).

Synthesis Example 7 Synthesis of 3me-coelenterazine (3me-CTZ)

Synthesis Example 7-1

The magnesium turnings (271 mg, 11.1 mmol) were dried in vacuo byheating with a heat gun. After cooling to room temperature and beingplaced under an argon atmosphere, THF (8 mL) was added thereto, followedby slow addition of 3-methylbenzyl chloride (71) (1.35 mL, 10.2 mmol) atroom temperature. The reaction mixture became warm as the result of anexothermic reaction and most of the magnesium turnings were consumed.After cooling to room temperature, it was used directly in the nextreaction as a THF solution of (3-methylbenzyl)magnesium chloride (72).

Under an argon atmosphere, to a solution of ethyl diethoxyacetate (1.80mL, 10.1 mmol) in THF (20 mL) was added slowly a THF solution of (72)prepared above at −78° C. After stirring at −78° C. for an hour, to thiswas added 20% aqueous solution of ammonium chloride (10 mL) and theproduct was extracted with ethyl acetate (×3). The organic layer wassequentially washed with water (×1) and saturated brine (×1), and driedover anhydrous sodium sulfate. After filtration, the mixture wasconcentrated under reduced pressure and the residue was purified bysilica gel flash column chromatography (n-hexane/ethyl acetate=9/1) togive 1,1-diethoxy-3-(3-methylphenyl)propan-2-one (73) as a colorlessoily substance (1.38 g, 5.84 mmol, 58.0%).

TLC R_(f)=0.40 (n-hexane/ethyl acetate=9/1);

¹H NMR (400 MHz, CDCl₃) δ 1.25 (t, 6H, J=7.0 Hz), 2.33 (s, 3H), 3.55(dq, 2H, J=9.5, 7.0 Hz), 3.70 (dq, 2H, J=9.5, 7.0 Hz), 3.85 (s, 2H),4.63 (s, 1H), 6.99-7.08 (m, 3H), 7.18-7.23 (m, 1H);

¹³C NMR (67.8 MHz, CDCl₃) δ 15.2 (2C), 21.4, 43.7, 63.4 (2C), 102.3,126.8, 127.6, 128.4, 130.6, 133.7, 138.1, 203.3;

IR (KBr, cm⁻¹) 700, 758, 1063, 1099, 1157, 1314, 1736, 2880, 2928, 2976;

HRMS (FAB⁺/NBA+NaI) m/z 259.1307 (M+Na, C₁₄H₂₀O₃Na required 259.1310).

Synthesis Example 7-2

Under an argon atmosphere, 1,1-diethoxy-3-(3-methylphenyl)propan-2-one(73) (265 mg, 1.12 mmol) was dissolved in 1,4-dioxane (2 mL) and water(0.4 mL). To this was added coelenteramine (202 mg, 728 μmol) and, aftercooling to 0° C., conc. hydrochloric acid (0.20 mL) was further addedthereto, and then stirred overnight (17 hours) at 100° C. After coolingto room temperature, the mixture was concentrated under reduced pressureand the residue was purified by silica gel flash chromatography in anargon flow (n-hexane/ethyl acetate→1/2→ethyl acetate→ethylacetate/methanol=50/1→20/1). The solid obtained was furtherreprecipitated (n-hexane/acetone) to give 3me-coelenterazine (74,3me-CTZ) as a yellow powder (148 mg, 351 μmol, 48.2%).

TLC R_(f)=0.52 (ethyl acetate/methanol=20/1);

HPLC retention time 17.1 min;

Mp 142-145° C. (dec.);

UV (MeOH) λ_(max) (ε)=260 (22500), 349.5 (5100), 435 (8900) nm;

FL (MeOH) λ_(max) Em. 437, 547 nm;

¹H NMR (400 MHz, CD₃OD) δ 2.29 (s, 3H), 4.24 (s, 2H), 4.54 (s, 2H),6.88-6.94 (AA′BB′, 2H), 7.03-7.14 (m, 3H), 7.16-7.21 (m, 1H), 7.22-7.34(m, 3H), 7.39-7.43 (m, 2H), 7.69-7.77 (AA′BB′, 2H), 8.30 (br, ¹H);

IR (KBr, cm⁻¹) 700, 748, 820, 841, 1171, 1236, 1277, 1506, 1541, 1589,1608, 1647, 2862, 2922, 3028;

HRMS (FAB⁺/glycerol) m/z 422.1863 (M+H, C₂₇H₂₄N₃O₂ required 422.1869).

Synthesis Example 8 Synthesis of 3meo-coelenterazine (3meo-CTZ)Synthesis Example 81

Under an argon atmosphere, 3-methoxyphenylacetyl chloride (81) (952 mg,5.16 mmol) was dissolved in THF (2.5 mL) and acetonitrile (2.5 mL) andcooled to 0° C. To this was slowly added a solution oftrimethylsilyldiazomethane in diethyl ether (2.0 M, 5.00 mL, 10.0 mmol),which was stirred overnight (15 hours) after warming up to roomtemperature. After concentrating under reduced pressure, the residue waspurified by silica gel flash column chromatography (n-hexane/diethylether=3/2) to give 1-diazo-3-(3-methoxyphenyl)propan-2-one (82) as apale yellow oily substance (650 mg, 3.42 mmol, 66.3%).

TLC R_(f)=0.38 (n-hexane/diethyl ether=1:2);

¹H NMR (400 MHz, CDCl₃) δ 3.59 (s, 2H), 3.81 (s, 3H), 5.14 (s, 1H),6.76-6.86 (m, 3H), 7.23-7.29 (m, 1H);

¹³C NMR (67.8 MHz, CDCl₃) δ 47.9, 54.7, 55.0, 112.6, 114.9, 121.6,129.7, 135.9, 159.8, 192.6;

IR (KBr, cm⁻¹) 691, 764, 876, 949, 1047, 1076, 1150, 1258, 1358, 1489,1584, 1631, 2104, 2835, 2940, 3003, 3580;

HRMS (EI) m/z 190.0740 (M, C₁₀H₁₀N₂O₂ required 190.0742).

Synthesis Example 8-2

Under an argon atmosphere, 1-diazo-3-(3-methoxyphenyl)propan-2-one (82)(501 mg, 2.63 mmol) was dissolved in anhydrous ethanol (5 mL) and cooledto −18° C. To this was added tert-butyl hypochlorite (300 μL, 2.65 mmol)and stirred for an hour at the same temperature. After concentratingunder reduced pressure, the residue was purified by silica gel flashcolumn chromatography (n-hexane/ethyl acetate=10/1) to give1,1-diethoxy-3-(3-methoxyphenyl)propan-2-one (83) as a pale yellow oilysubstance (371 mg, 1.47 mmol, 55.8%).

TLC R_(f)=0.27 (n-hexane/ethyl acetate=9/1);

¹H NMR (400 MHz, CDCl₃) δ 1.25 (t, 6H, J=7.0 Hz), 3.55 (dq, 2H, J=9.5,7.0 Hz), 3.70 (dq, 2H, J=9.5, 7.0 Hz), 3.79 (s, 3H), 3.86 (s, 2H), 4.64(s, 1H), 6.74-6.83 (m, 3H), 7.21-7.25 (m, 1H);

¹³C NMR (67.8 MHz, CDCl₃) δ 15.2 (2C), 43.8, 55.2, 63.4 (2C), 102.3,112.5, 115.4, 122.2, 129.4, 135.2, 159.7, 203.1;

IR (KBr, cm⁻¹) 1057, 1098, 1150, 1260, 1585, 1732, 2835, 2886, 2934,2976;

HRMS (FAB⁺/NBA+KCl) m/z 291.0992 (M+K, C₁₄H₂₀O₄K required 291.0999).

Synthesis Example 8-3

Under an argon atmosphere, 1,1-diethoxy-3-(3-methoxyphenyl)propan-2-one(83) (254 mg, 1.01 mmol) was dissolved in 1,4-dioxane (2 mL) and water(0.4 mL). To this was added coelenteramine (209 mg, 754 μmol) and, aftercooling to 0° C., conc. hydrochloric acid (0.20 mL) was further addedthereto, and then stirred overnight (15 hours) at 100° C. After coolingto room temperature, the mixture was concentrated under reduced pressureand the residue was purified by silica gel flash chromatography in anargon flow (n-hexane/ethyl acetate=1/2→ethyl acetate→ethylacetate/methanol=20/1→10/1) (×2). The solid obtained was furtherreprecipitated (n-hexane/acetone) to give 3meo-coelenterazine (84,3meo-CTZ) as an ocher powder (98.7 ng, 226 μmol, 29.9%).

TLC R_(f)=0.33 (ethyl acetate/methanol=20/1):

HPLC retention time 13.8 min;

Mp 136-140° C. (dec.);

UV (MeOH) λ_(max) (ε)=262 (25600), 346.5 (5400), 438.5 (9900) nm;

FL (MeOH) λ_(max) Em. 428.5, 543.5 nm;

¹H NMR (400 MHz, CD₃OD) δ 3.75 (s, 3H), 4.25 (s, 2H), 4.55 (s, 2H), 6.77(dd, 1H, J=1.9, 8.0 Hz), 6.84-6.92 (m, 4H), 7.16-7.34 (m, 4H), 7.38-7.44(m, 2H), 7.63-7.70 (AA′BB′, 2H), 8.17 (br, ¹H);

IR (KBr, cm⁻¹) 700, 750, 839, 1045, 1170, 1260, 1506, 1541, 1595, 1608,1647, 2835, 2938, 3030, 3059;

HRMS (FAB⁺/glycerol) m/z 438.1814 (M+H, C₂₇H₂₄N₃O₃ required 438.1818).

Synthesis Example 9 Synthesis of αmeh-coelenterazine (αmeh-CTZ)

Synthesis Example 9-1

The magnesium turnings (274 mg, 11.3 mmol) were dried in vacuo byheating with a heat gun. After cooling to room temperature and beingplaced under an argon atmosphere, THF (8 mL) was added thereto, followedby slow addition of 1-chloroethylbenzene (91) (1.35 mL, 10.2 mmol) atroom temperature. The reaction mixture became warm as the result of anexothermic reaction and most of magnesium turnings were consumed. Aftercooling to room temperature, it was used directly in the next reactionas a THF solution of (1-phenylethyl)magnesium chloride (92).

Under an argon atmosphere, to a solution of ethyl diethoxyacetate (1.80mL, 10.1 mmol) in THF (20 mL) was added slowly a THF solution of (92)prepared above at −78° C. After stirring at −78° C. for 3 hours, to thiswas added 20% aqueous solution of ammonium chloride (10 mL) and theproduct was extracted with ethyl acetate (×3). The organic layer wassequentially washed with water (×1) and saturated brine (×1), and driedover anhydrous sodium sulfate. After filtration, the mixture wasconcentrated under reduced pressure and the residue was purified bysilica gel flash column chromatography (n-hexane/ethyl acetate=19/1) togive 1,1-diethoxy-3-phenylbutan-2-one (93) as a colorless oily substance(330 mg, 1.37 mmol, 13.7%).

TLC R_(f)=0.43 (n-hexane/ethyl acetate=9/1);

¹H NMR (400 MHz, CDCl₃)□ δ 1.13 (t, 3H, J=7.0 Hz), 1.20 (t, 3H, J=7.0Hz), 1.41 (d, 3H, J=7.0 Hz), 3.32 (dq, 1H, J=9.5, 7.0 Hz), 3.47-3.64 (m,3H), 4.26 (q, 1H, J=7.0 Hz), 4.59 (s, 1H), 7.22-7.36 (m, 5H);

¹³C NMR (67.8 MHz, CDCl₃) δ 15.1, 15.2, 18.1, 47.3, 62.8, 63.0, 101.2,127.1, 128.3 (2C), 128.7 (2C), 140.2, 205.8;

IR (KBr, cm⁻¹) 700, 1030, 1063, 1103, 1163, 1452, 1493, 1730, 2874,2932, 2976;

HRMS (FAB⁺/NBA+NaI) m/z 237.1496 (M+H, C₁₄H₂₁O₃ required 237.1491).

Synthesis Example 9-2

Under an argon atmosphere, 1,1-diethoxy-3-phenylbutan-2-one (93) (188mg, 796 μmol) was dissolved in 1,4-dioxane (2 mL) and water (0.4 mL). Tothis was added coelenteramine (170 mg, 613 μmol) and, after cooling to0° C., conc. hydrochloric acid (0.20 mL) was further added thereto, andthen stirred overnight (16 hours) at 100° C. After cooling to roomtemperature, the mixture was concentrated under reduced pressure and theresidue was purified by silica gel flash chromatography in an argon flow(n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).The solid obtained was further reprecipitated (n-hexane/acetone) to giveαmeh-coelenterazine (94, αmeh-CTZ) as a yellow powder (76.5 mg, 181μmol, 29.6%).

TLC R_(f)=0.53 (ethyl acetate/methanol=20/1);

HPLC retention time 17.6 min;

Mp 143-145° C. (dec.);

UV (MeOH) λ_(max) (ε)=259.5 (18800), 350 (4400), 439 (7800) nm;

FL (MeOH) λ_(max) Em. 429.5, 551 nm;

¹H NMR (400 MHz, CD₃OD) δ 1.81 (d, 3H, J=7.3 Hz), 4.56 (q, 1H, J=7.3Hz), 4.58 (s, 2H), 6.89-6.94 (AA′BB′, 2H), 7.18-7.40 (m, 8H), 7.43-7.47(m, 2H), 7.58-7.65 (AA′BB′, 2H), 8.07 (br, ¹H);

IR (KBr, cm⁻¹) 700, 820, 841, 1177, 1215, 1277, 1454, 1508, 1541, 1558,1610, 1647, 2876, 2934, 2972, 3030, 3059:

HRMS (FAB⁺/glycerol) m/z 422.1872 (M+H, C₂₇H₂₄N₃O₂ required 422.1869).

Syntheses Example 10 Synthesis of 3-isocoelenterazine (3iso-CTZ)

Synthesis Example 10-1

3-(tert-Butyldimethylsilyloxy)benzyl alcohol (101) (prepared by theprocess described in Wu, Y.-C. et al., J. Am. Chem. Soc., 130, 7148-7152(2008)) (7.94 g, 31.4 mmol) was dissolved in dichloromethane (150 mL),and cooled to 0° C. To this were sequentially added triethylamine (9.10mL, 66.7 mmol) and methanesulfonyl chloride (3.80 mL, 49.1 mmol) andstirred for 25 hours after warming up to room temperature. To this wasadded water, and the product was extracted with dichloromethane (×3).The organic layer was sequentially washed with water (×1) and saturatedbrine (×1), and dried over anhydrous sodium sulfate. After filtration,the mixture was concentrated under reduced pressure and the residue waspurified by silica gel flash column chromatography (n-hexane/ethylacetate=95/1) to give 3-(tert-butyldimethylsilyloxy)benzyl chloride(102) as a colorless oily substance (5.90 g, 23.0 mmol, 73.1%).

TLC R_(f)=0.45 (n-hexane);

¹H NMR (400 MHz, CDCl₃) δ 0.20 (s, 6H), 0.99 (s, 9H), 4.53 (s, 2H), 6.79(dd, 1H, J=1.4, 8.1 Hz), 6.87 (d, 1H, J=1.4 Hz), 6.97 (d, 1H, J=8.1 Hz),7.20 (dd, 1H, J=8.1, 8.1 Hz).

Synthesis Example 10-2

The magnesium turnings (270 mg, 11.1 mmol) were dried in vacuo byheating with a heat gun. After cooling to room temperature and beingplaced under an argon atmosphere, THF (17.5 mL) was added thereto,followed by slow addition of 3-(tert-Butyldimethylsilyloxy)benzylchloride (102) (2.57 g, 10.0 mmol) at room temperature. The reactionmixture was warmed as the result of an exothermic reaction and most ofthe magnesium turnings were consumed. After cooling to room temperature,it was used directly in the next reaction as a THF solution of3-(tert-butyldimethylsilyloxy)benzylmagnesium chloride (103) was cooledto room temperature and was used as it was in the next reaction.

Under an argon atmosphere, to a solution of ethyl diethoxyacetate (1.80mL, 10.1 mmol) in THF (20 mL) was added slowly a THF solution of (103)prepared above at −78° C. After stirring at −78° C. for an hour, to thiswas added 20% aqueous solution of ammonium chloride (30 mL) and theproduct was extracted with ethyl acetate (×3). The organic layer wassequentially washed with water (×1) and saturated brine (×1), and driedover anhydrous sodium sulfate. After filtration, the mixture wasconcentrated under reduced pressure and the residue was purified bysilica gel flash column chromatography (n-hexane/ethyl acetate=10/1) togive 3-[3-(tert-butyldimethylsilyloxy]phenyl]-1,1-diethoxypropan-2-one(104) as a colorless oily substance (1.90 g, 5.39 mmol, 53.9%).

TLC R_(f)=0.48 (n-hexane/ethyl acetate=10/1);

¹H NMR (400 MHz, CDCl₃) δ 0.19 (s, 6H), 0.97 (s, 9H), 1.25 (t, 6H, J=7.0Hz), 3.54 (dq, 2H, J=9.5, 7.0 Hz), 3.69 (dq, 2H, J=9.5, 7.0 Hz), 3.82(s, 2H), 4.63 (s, 1H), 6.68-6.75 (m, 2H), 6.81 (d, 1H, J=7.8 Hz), 7.16(dd, 1H, J=7.8, 7.8 Hz);

¹³C NMR (67.8 MHz, CDCl₃) δ −4.4 (2C), 15.2 (2C), 18.2, 25.7 (3C), 43.7,63.3 (2C), 102.2, 118.5, 121.6, 122.8, 129.3, 135.2, 155.7, 202.9;

IR (KBr, cm⁻¹) 781, 839, 982, 1063, 1157, 1275, 1487, 1585, 1601, 1736,2859, 2886, 2930, 2955, 2974;

HRMS (EI) nm/z 352.2073 (M, C₁₉H₃₂O₄Si required 352.2070).

Synthesis Example 10-3

Under an argon atmosphere,3-[3-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (104)(390 mg, 1.11 mmol) was dissolved in 1,4-dioxane (2 mL) and water (0.4mL). To this was added coelenteramine (202 mg, 728 μmol) and, aftercooling to 0° C., conc. hydrochloric acid (0.20 mL) was added thereto,and then stirred overnight (14 hours) at 100° C. After cooling to roomtemperature, the mixture was concentrated under reduced pressure and theresidue was purified by silica gel flash chromatography in an argon flow(n-hexane/ethyl acetate 1/2→ethyl acetate→ethyl acetate/methanol=20/1).The solid obtained was further reprecipitated (n-hexane/acetone) to give3-isocoelenterazine (105, 3iso-CTZ) as a yellow powder (159 mg, 375μmol, 51.5%).

TLC R_(f)=0.29 (ethyl acetate/methanol=20/1);

HPLC retention time 8.6 min;

Mp 160-162° C. (dec.);

UV (MeOH) λ_(max) (ε)=265.5 (19300), 351.5 (4400), 433.5 (7700) nm;

FL (MeOH) λ_(max) Em. 429, 549.0 nm;

¹H NMR (400 MHz, CD₃OD) δ 4.20 (s, 2H), 4.53 (s, 2H), 6.66 (dd, 1H,J=1.4, 8.1 Hz), 6.71-6.77 (m, 2H), 6.88-6.93 (AA′BB′, 2H), 7.12 (dd, 1H,J=8.1, 8.1 Hz), 7.22-7.35 (m, 3H), 7.39-7.43 (m, 2H), 7.68-7.75 (AA′BB′,2H), 8.26 (br, ¹H);

IR (KBr, cm⁻¹) 700, 760, 820, 841, 1171, 1238, 1275, 1456, 1506, 1541,1591, 1608, 2953, 3063, 3150;

HRMS (FAB⁺/glycerol) m/z 424.1663 (M+H, C₂₆H₂₂N₃O₃ required 424.1661).

Comparative Synthesis Example 1 Synthesis of i-coelenterazine (i-CTZ)

Comparative Synthesis Example 1-1

Under an argon atmosphere, to 4-iodophenylacetic acid (11) (prepared bythe process described in Chen, Q.-H. et al., Bioorg. Med. Chem., 14,7898-7909 (2006)) (1.06 g, 4.05 mmol) was added thionyl chloride (5.00mL, 68.6 mmol) and heated to reflux (100° C.) for 1.5 h. After coolingto room temperature, the mixture was concentrated under reduced pressureto give 4-iodophenylacetyl chloride (12) as a brown oily crude product.

Under an argon atmosphere, 4-iodophenylacetyl chloride (12) obtainedabove was dissolved in THF (2 mL) and acetonitrile (2 mL) and cooled to0° C. To this was slowly added a solution of trimethylsilyldiazomethanein diethyl ether (2.0 M, 4.00 mL, 8.00 mmol), which was stirredovernight (14 h) after warming up to room temperature. Afterconcentrating under reduced pressure, the residue was purified by silicagel flash column chromatography (n-hexane/diethyl ether=1/1) to give1-diazo-3-(4-iodophenyl)propan-2-one (13) as a pale yellow solid (635mg, 4.44 mmol, 55.5%, 2 steps).

Comparative Synthesis Example 1-2

1-Diazo-3-(4-iodophenyl)propan-2-one (13) (1.27 g, 4.43 mmol) wasdissolved in acetic acid (20 mL) and cooled to 0° C. To this was added47% hydrobromic acid (1.55 mL, 13.3 mmol) and stirred for an hour afterwarming to room temperature. After neutralization by adding saturatedaqueous solution of sodium bicarbonate, the product was extracted withethyl acetate (×3) and the organic layer was washed sequentially withsaturated aqueous solution of sodium bicarbonate (×1) and saturatedbrine (×1), and dried over anhydrous sodium sulfate. After filtration,the mixture was concentrated under reduced pressure and the residue waspurified by silica gel flash column chromatography (n-hexane/ethylacetate=7/1) to give 1-bromo-3-(4-iodophenyl)propan-2-one (16) as acolorless solid (1.42 g, 4.18 mmol, 94.4%).

TLC R_(f)=0.41 (n-hexane/ethyl acetate=7/1);

¹H NMR (400 MHz, CDCl₃) δ 3.90 (s, 2H), 3.91 (s, 2H), 6.96-7.01 (AA′BB′,2H), 7.66-7.71 (AA′BB′, 2H).

Comparative Synthesis Example 1-3

1-Bromo-3-(4-iodophenyl)propan-2-one (16) (460 mg, 1.36 mmol) wasdissolved in acetonitrile (4 mL) and to this was added silver nitrate(596 mg, 3.51 mmol) dissolved in acetonitrile (4 mL), and stirredovernight (13 h) at room temperature. The reaction solution was filteredthrough Celite and the filtrate was washed sequentially with water (×1)and saturated brine (×1), and then the organic layer was dried overanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure to give 3-(4-iodophenyl)-2-oxopropylnitrate (17) as a colorless solid (490 mg, <1.36 mmol, <100%), which wasused in the next reaction without further purification.

TLC R_(f)=0.26 (tailing) (n-hexane/ethyl acetate=5/1);

¹H NMR (400 MHz, CDCl₃) δ 3.73 (s, 2H), 4.97 (s, 2H), 6.96-7.00 (AA′BB′,2H), 7.68-7.73 (AA′BB′, 2H).

Comparative Synthesis Example 1-4

3-(4-Iodophenyl)-2-oxopropyl nitrate (17) (450 mg) was dissolved indimethylsulfoxide (20 mL) and to this was added sodium acetatetrihydrate (211 mg, 1.55 mmol), and stirred for an hour at roomtemperature. To this was added water and the product was extracted withdiethyl ether (×3). The organic layer was washed sequentially with water(×1), saturated aqueous solution of sodium bicarbonate (×1) andsaturated brine (×1), and then dried over anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressureto give 3-(4-iodophenyl)-2-oxopropanal (18) as an orange solid (346 mg,<1.26 mmol, <90.1%), which was used in the next reaction without furtherpurification.

TLC R_(f)=0.43 (tailing) (n-hexane/ethyl acetate=5/1);

Comparative Synthesis Example 1-5

Under an argon atmosphere, 3-(4-iodophenyl)-2-oxopropanal (18) (343 mg,1.25 mmol) was dissolved in 1,4-dioxane (2.0 mL) and water (0.4 mL). Tothis was added coelenteramine (233 mg, 834 μmol) and, after cooling to0° C., conc. hydrochloric acid (0.20 mL) was added thereto, and thenstirred for 6 h at 100° C. After cooling to room temperature, themixture was extracted with dichloromethane and a small quantity ofmethanol (×4). The organic layer was washed sequentially with water (×1)and saturated brine (×1) and then dried over anhydrous sodium sulfate.After filtration, the organic layer was concentrated under reducedpressure and the residue was purified in an argon flow by silica gelflash chromatography (deaerated dichloromethane/deaeratedmethanol=50/1→10/1). The solid obtained was further reprecipitated(n-hexane/acetone) to give i-coelenterazine (15, i-CTZ) as an ocherpowder (52.6 mg, 98.6 μmol, 11.8%).

n-CTZ, i-CTZ, me-CTZ, et-CTZ, cf3-CTZ, meo-CTZ, 3me-CTZ, 3meo-CTZ,αmeh-CTZ and 3iso-CTZ prepared above were used for the production ofsemi-synthetic aequorins and for the analysis of substrate specificityof each luciferase, and so on, in the following EXAMPLES. In addition,coelenterazine (CTZ) and h-coelenterazine (h-CTZ) were used in thefollowing EXAMPLES. CTZ and h-CTZ used were those manufactured by ChissoCorporation.

Hereinafter, CTZ, or h-CTZ, n-CTZ, me-CTZ, et-CTZ, cf3-CTZ, meo-CTZ,3me-CTZ, 3meo-CTZ, αmeh-CTZ, 3iso-CTZ or i-CTZ is sometimes referred toas coelenterazine or analogs thereof.

Example 1 Production of Semi-Synthetic Aequorins for SubstrateSpecificity Analysis and Measurement of Luminescence Activity

To produce the following semi-synthetic aequorins, the recombinantapoaequorin manufactured by Chisso Corporation was used. Thisrecombinant apoaequorin was obtained by expressing and purifyingaccording to the method described in Inouye, S. and Sahara, Y. ProteinExpress. Purif. (2007) 53: 384-389, using piP-H-HE constructed from theexpression vector piP-HEΔE, which is described in the same literature byinserting histidine sequence therein.

(1) Production of Semi-Synthetic Aequorins

One microliter of 2-mercaptoethanol and 1.31 μg of recombinantapoaequorin solution (made by Chisso Corp.) were added to and mixed with1 ml of 30 mM Tris-HCl (pH 7.6) containing 10 mM EDTA. Subsequently, 1μl of a solution of coelenterazine or its analog in ethanol was addedand the mixture was allowed to stand at 4° C. to convert into asemi-synthetic aequorin. In order to confirm the regeneration time andregeneration efficiency from apoaequorin to the semi-synthetic aequorin,the luminescence activity was assayed at the respective points of theregeneration process (at the respective points of 0.5, 1, 1.5, 2, 3 and18 hours from the start of regeneration). The results are shown in FIG.1.

As shown in FIG. 1, it is demonstrated that coelenterazine analogs ofthe present invention (me-CTZ, et-CTZ, cf3-CTZ, meo-CTZ, 3me-CTZ,3meo-CTZ, αmeh-CTZ and 3iso-CTZ) can be luminescence substrates forsemi-synthetic aequorins, though luminescent intensities are different.

2) Method for Measuring the Luminescence Activity

Specifically, the luminescence activity described above was measured asfollows. The luminescence reaction was started by adding 100 μl of 50 mMTris-HCl (pH 7.6) containing 50 mM calcium chloride solution to 2 μl ofa solution of the semi-synthetic aequorin at each regeneration process.The luminescence activity was measured for 10 seconds with a luminometerLuminescencer-PSN AB2200 (manufactured by Atto Co., Ltd.). The measuredvalues were represented as the maximum intensity (I_(max)) ofluminescence. The emission for 60 seconds was integrated, which was madea luminescence capacity.

Example 2 Method for Measuring the Luminescence Patterns and Half DecayTime of Semi-Synthetic Aequorins

A solution of the regenerated semi-synthetic aequorin was diluted to10-fold with 20 mM Tris-HCl (pH 7.6) containing 0.1% BSA (manufacturedby Sigma), 0.01 mM EDTA and 150 mM NaCl. The dilution was dispensed intoa 96-well microplate (Nunc #236108) in 5 μl/well. The luminescencereaction was started by injecting 100 μl/well of 50 mM Tris-HCl (pH 7.6)containing 50 mM calcium chloride solution using a luminescence platereader Centro LB960 (manufactured by Berthold). The luminescencepatterns for 60 seconds were measured to determine the half decay timeof luminescence (time period to reach the half of the maximumluminescence intensity). The results are shown in FIG. 2 and summarizedin TABLE 1 below.

TABLE 1 Luminescence properties of semi-synthetic aequorins Half MaximumLuminescence Luminescence decay emission Coelenterazine activitycapacity time wavelength derivative I_(max) (%) 60 sec (%) sec. λ_(max)(nm) Coelenterazine 100.0 100.0 0.81 465.5 h- 87.9 70.1 0.69 465.5 et-27.3 44.1 1.21 472.0 n- 3.6 24.0 4.71 467.0 cf3- 6.3 48.6 5.95 473.5 i-5.0 65.3 11.42 472.0 meo- 22.3 60.1 1.63 469.0 3meo- 64.9 35.6 0.52466.0 me- 5.8 45.0 5.00 469.5 3me- 35.0 21.2 0.51 466.0 αmeh- 2.2 0.90.35 467.5 3iso- 51.6 13.4 0.24 464.0

As shown in FIG. 2 and TABLE 1, the half decay time of luminescence waslonger than CTZ especially with the semi-synthetic aequorins usingme-CTZ and cf3-CTZ as the luminescence substrates. Accordingly, Ca²⁺ canbe measured by light being allowed to be emitted slowly, not byinstantaneous emission. This indicates that the semi-synthetic aequorinswherein me-CTZ and cf3-CTZ are used as the luminescence substrates arewell suited for applications to a high-precision assay system using asan indicator Ca²⁺ level change in the system, in the same manner as inthe semi-synthetic aequorins wherein i-CTZ and n-CTZ conventionallyknown as aequorins having slow half decay time are used as theluminescence substrates.

Example 3 Method for Measuring the Emission Spectra of Semi-SyntheticAequorins

In a quartz cell with an optical path length of 10 mm, 1 ml of 50 mMTris-HCl (pH 7.6) containing 1 mM EDTA and 100 μl (100 μg protein) of asolution of the regenerated semi-synthetic aequorin were charged andthey were mixed. Subsequently, 100 μl of 50 mM Tris-HCl (pH 7.6)containing 0.1 ml of 10 mM calcium chloride solution was added to themixture to trigger the luminescence reaction. The spectra were measuredon a spectrofluorimeter (FP-6500, manufactured by JASCO Corporation)with the excitation source turned off. The measurement conditions usedwere as follows: band width, 20 nm; response, 0.5 second and scan speed,2000 nm/min at 22 to 25° C. The results are shown in FIG. 3. In FIG. 3,AQ denotes semi-synthetic aequorin, for which coelenterazine is theluminescence substrate.

As shown in FIG. 3, the semi-synthetic aequorins, for which me-CTZ,et-CTZ, cf3-CTZ, meo-CTZ, 3me-CTZ, 3meo-CTZ and 3iso-CTZ are theluminescence substrates exhibit different emission spectra from those ofknown coelenterazine analogs (h-CTZ and i-CTZ).

Example 4 Preparation of Standard Calcium Solution

By dissolving 1 ml of 1 g/L standard calcium carbonate solution(manufactured by Wako Pure Chemical Industry) in 9 ml of 50 mM Tris-HCl(pH 7.6), 10⁻³ M calcium carbonate solution was prepared.

One milliliter of the resulting 10⁻³ M calcium carbonate solution wastaken and added to 9 ml of 50 mM Tris-HCl (pH 7.6) to prepare 10⁻⁴Mcalcium carbonate solution. Furthermore, 3 ml of the resulting 10⁻⁴Mcalcium carbonate solution was taken and added to 6 ml of 50 mM Tris-HCl(pH 7.6) to prepare 3×10⁻⁴ M calcium carbonate solution. Next, 1 ml ofthe resulting 10⁻⁴ M calcium carbonate solution was taken and added to 9ml of 50 mM Tris-HCl (pH 7.6) to prepare 10⁻⁵ M calcium carbonatesolution. Further 3 ml of the resulting 10⁻⁵ M calcium carbonatesolution was taken and 6 ml of 50 mM Tris-HCl (pH 7.6) was added toprepare 3×10⁻⁵ M calcium carbonate solution. The dilution series wasprepared by the serial process above to prepare standard calciumsolution of 10⁻³ M to 10⁻⁸ M.

Example 5 Production of Semi-Synthetic Aequorin for Detecting CalciumLevels

After 5 mg of the recombinant apoaequorin (manufactured by Chisso Corp.)was dissolved in 5 ml of 50 mM Tris-HCl (pH 7.6) containing 10 mM DTTand 30 mM EDTA, 100 μg of a solution of 1.2-fold equivalent ofcoelenterazine analog in ethanol was added thereto. The mixture wasallowed to stand at 4° C. overnight to convert into the semi-syntheticaequorin. The semi-synthetic aequorin obtained was concentrated usingAmicon Ultra-4 (manufactured by Millipore, molecular weight cut off,10.000). Subsequently, the concentrate was washed 3 times with 3 ml of30 mM Tris-HCl (pH 7.6) containing 0.05 mM EDTA to remove an excess ofcoelenterazine analog and make the EDTA concentration 0.05 mM.

This semi-synthetic aequorin solution (2.5 mg/ml) was diluted with 20 mMTris-HCl (pH 7.6) containing 0.1% BSA (manufactured by Sigma), 0.01 mMEDTA and 150 mM NaCl.

Example 6 Preparation of Calcium Standard Curve

The calcium standard solution prepared as described above was dispensedinto a 96-well microplate (Nunc #236108) in 50 μl/well, and thesemi-synthetic aequorin solution diluted was injected in 10 id/well. Theluminescence intensity was measured for 60 seconds using a luminescenceplate reader Centro LB960 (manufactured by Berthold) and expressed interms of the maximum luminescence intensity (I_(max)). The luminescenceintensity was measured for each semi-synthetic aequorin in the samefashion. Based on the maximum luminescence intensity (I_(max)) obtained,the calcium standard curve for each semi-synthetic aequorin wasprepared.

The results are shown in FIG. 4.

As shown in FIG. 4, it is observed that the photoproteins of the presentinvention can be used for the detection, quantification or the like ofcalcium ions, since the calcium standard curves can be prepared by usingthe semi-synthetic aequorins, which were prepared from coelenterazineanalogs of the present invention (me-CTZ, et-CTZ, cf3-CTZ, meo-CTZ,3me-CTZ, 3meo-CTZ, αmeh-CTZ and 3iso-CTZ).

Example 7 Method for Analyzing the Substrate Specificity and Measuringthe Luminescence Activity of the 19 kDa Protein from OplophorusLuciferase

The 19 kDa protein of Oplophorus luciferase was purified by the methoddescribed in Inouye, S. and Sasaki, S. Protein Express. and Purif (2007)56: 261-268, which was provided for use.

After 1 μl of the 19 kDa protein (2.3 mg/ml) of Oplophorus luciferasecontaining 1 mM DTT was dissolved in 100 μl of 30 mM Tris-HCl (pH 7.6)containing 10 mM EDTA, 1 μl of a solution of coelenterazine or itsanalog in ethanol (1 μg/μl) was mixed with the solution to trigger theluminescence reaction. The luminescence activity was measured for 60seconds using a luminometer Luminescencer-PSN AB2200 (manufactured byAtto Co., Ltd.). The luminescence activity was measured 3 times andexpressed as the maximum intensity (I_(max)) of luminescence. Theemission for 10 seconds was integrated, which was made a luminescencecapacity.

The emission spectra were assayed by adding 5 μg of coelenterazine orits analog (dissolved in 5 μl of ethanol) to 990 μl of 30 mM Tris-HCl(pH 7.6) containing 10 mM EDTA and then adding 20 μl (46 μg) ofOplophorus luciferase thereby to trigger luminescence. The spectra weremeasured on a spectrofluorimeter (FP-6500, manufactured by JASCOCorporation) with the excitation source turned off, under band width of20 nm, response of 0.5 second and scan speed of 2000 nm/min at 22 to 25°C. The results of luminescence activity, luminescence capacity andmaximum emission wavelength are shown in TABLE 2 below. The emissionspectrum charts are shown in FIG. 5.

TABLE 2 Substrate specificity and maximum emission wavelength ofOplophorus luciferase using coelenterazine derivatives MaximumLuminescence Luminescence emission Coelenterazine activity capacitywavelength analog I_(max) (%) 10 secs. (%) λ_(max) (nm) Coelenterazine100.0 100.0 457.0 h- 68.4 110.2 456.5 i- 32.3 53.8 450.0 et- 21.1 39.5454.0 cf3- 49.5 68.2 450.5 me- 46.6 82.7 454.4 3me- 80.0 122.8 455.5meo- 68.1 108.6 454.5 3meo- 189.1 204.7 455.5 αmeh- 15.6 19.0 457.03iso- 78.2 71.8 458.0

As shown in TABLE 2, it is observed that me-CTZ, et-CTZ, cf3-CTZ,meo-CTZ, 3me-CTZ, 3meo-CTZ, αme-CTZ and 3iso-CTZ become relatively goodluminescence substrates for Oplophorus luciferase. Especially, 3me-CTZ,3meo-CTZ and 3iso-CTZ exhibit high luminescence activity and/orluminescence capacity when compared to known h-CTZ.

Example 8 Method for Analyzing the Substrate Specificity and Measuringthe Luminescence Activity of Gaussia Luciferase

Gaussia luciferase was purified by the method described in JPA2008-099669 and provided for use.

After 1 μl of Gaussia luciferase (0.16 mg/ml) was dissolved in 100 μl ofphosphate buffered saline (manufactured by Sigma Inc.) containing 0.01%Tween 20 (manufactured by Sigma Inc.) and 10 mM EDTA, 1 μl of a solutionof coelenterazine or its analog dissolved in ethanol (1 μg/μl) was mixedwith the solution to trigger the luminescence reaction. The luminescenceactivity was measured for 10 seconds using a luminometerLuminescencer-PSN AB2200 (manufactured by Atto Co., Ltd.). Theluminescence activity was measured 3 times and expressed as the maximumintensity (I_(max)) of luminescence. The emission spectra were assayedby adding 5 μg of coelenterazine or its analog (dissolved in 5 μl ofethanol) to 1000 μl of phosphate buffered saline containing 10 mM EDTAand 0.01% Tween 20, and then adding 1 μl (0.23 μg) of Gaussia luciferasethereby to trigger luminescence. The spectra were measured using aspectrofluorimeter (FP-6500, manufactured by JASCO Corporation) with theexcitation source turned off, under band width of 20 nm, response of 0.5second and scan speed of 2000 nm/min of 20 nm, response of 0.5 secondand scan speed of 2000 nm/min at 22 to 25° C. The results ofluminescence activity, luminescence capacity and maximum emissionwavelength are shown in TABLE 3 below. The emission spectrum charts areshown in FIG. 6.

TABLE 3 Substrate specificity and maximum emission wavelength of Gaussialuciferase using coelenterazine derivatives Maximum LuminescenceLuminescence emission Coelenterazine activity capacity wavelength analogI_(max) (%) 10 secs. (%) λ_(max) (nm) Coelenterazine 100.0 100.0 473.0h- 5.8 7.2 474.0 i- 0.9 1.3 475.0 et- 0.9 1.1 476.0 cf3- 0.8 0.7 476.5me- 4.5 5.9 475.0 3me- 2.6 2.1 475.5 meo- 13.6 15.5 475.5 3meo- 3.8 3.1474.5 αmeh- >0.01 >0.01 473.0 3iso- 14.3 7.4 477.0

Gaussia luciferase exhibits high substrate specificity, and anycoelenterazine analog which acts as its luminescence substrate isunknown to date. As shown in TABLE 3, it is demonstrated that me-CTZ and3iso-CTZ become effective luminescence substrates for Gaussialuciferase.

Example 9 Method for Analyzing the Substrate Specificity of RenillaLuciferase and Measuring the Luminescence Activity

Renilla luciferase was purified by the method described in Inouye, S. &Shimomura, O. Biochem. Biophys. Res. Commun. (1997) 233: 349-353, whichwas provided for use.

After 1 μl of Renilla luciferase (0.45 mg/ml) was dissolved in 100 μl of30 mM Tris-HCl (pH 7.6) containing 10 mM EDTA, 1 μl of a solution ofcoelenterazine or its analog in ethanol (1 μg/μl) was mixed with thesolution to trigger the luminescence reaction. The luminescence activitywas measured 3 times using a luminometer Luminescencer-PSN AB2200(manufactured by Atto Co., Ltd.) and expressed as the maximum intensity(I_(max)) of luminescence. Emission was started by adding 5 μg ofcoelenterazine or its analog (dissolved in 5 μl of ethanol) to 1000 μlof 30 mM Tris-HCl (pH 7.6) containing 10 mM EDTA and 5 μl (2.3 μg) ofRenilla luciferase. The emission spectra were measured on aspectrofluorimeter (FP-6500, manufactured by JASCO Corporation) with theexcitation source turned off under band width of 20 nm, response of 0.5second and scan speed of 2000 nm/min at 22 to 25° C. The results ofluminescence activity, luminescence capacity and maximum emissionwavelength are shown in TABLE 4 below. The emission spectrum charts areshown in FIG. 7.

TABLE 4 Substrate specificity and maximum emission wavelength of Renillaluciferase using coelenterazine derivatives Maximum LuminescenceLuminescence emission Coelenterazine activity capacity wavelength analogI_(max) (%) 10 secs. (%) λ_(max) (nm) Coelenterazine 100.0 100.0 472.5h- 78.6 90.3 472.5 i- 0.2 0.3 473.0 et- 0.5 0.3 471.0 cf3- 0.8 0.8 474.5me- 6.6 7.0 472.0 3me- 12.7 13.1 471.5 meo- 8.0 8.6 472.5 3meo- 26.031.6 473.5 αmeh- 0.0 0.0 N.D.* 3iso- 25.7 27.5 472.0 N.D.: not detected

As shown in TABLE 4, it is demonstrated that me-CTZ, meo-CTZ, 3meo-CTZand 3iso-CTZ can be substrates for Renilla luciferase.

[Sequence Listing Free Text]

[SEQ ID NO: 1] Nucleotide sequence of natural apoaequorin[SEQ ID NO: 2] Amino acid sequence of natural apoaequorin[SEQ ID NO: 3] Nucleotide sequence of natural apoclytin-I[SEQ ID NO: 4] Amino acid sequence of natural apoclytin-I[SEQ ID NO: 5] Nucleotide sequence of natural apoclytin-II[SEQ ID NO: 6] Amino acid sequence of natural apoclytin-II[SEQ ID NO: 7] Nucleotide sequence of natural apomitrocomin[SEQ ID NO: 8] Amino acid sequence of natural apomitrocomin[SEQ ID NO: 9] Nucleotide sequence of natural apobelin[SEQ ID NO: 10] Amino acid sequence of natural apobelin[SEQ ID NO: 11] Nucleotide sequence of natural apobervoin[SEQ ID NO: 12] Amino acid sequence of natural apobervoin[SEQ ID NO: 13] Nucleotide sequence of Renilla luciferase[SEQ ID NO: 14] Amino acid sequence of Renilla luciferase[SEQ ID NO: 15] Nucleotide sequence of Oplophorus luciferase[SEQ ID NO: 16] Amino acid sequence of Oplophorus luciferase[SEQ ID NO: 17] Nucleotide sequence of Gaussia luciferase[SEQ ID NO: 18] Amino acid sequence of Gaussia luciferase

1-33. (canceled)
 34. A method for measuring a transcription activity ordetecting an analyte, which comprises using a luciferase derived fromRenilla sp., Oplophorus sp., or Gaussia sp., and a compound representedby general formula (1) below:

wherein: R¹ is (a) hydrogen, (b) hydroxy, (c) an alkyl having 1 to 4carbon atoms which may optionally be substituted with an alicyclicgroup, or (d) trifluoromethyl; R² is (a) hydrogen, (b) hydroxy, (c) ahalogen, (d) an alkyl having 1 to 4 carbon atoms which may optionally besubstituted with an alicyclic group, (e) trifluoromethyl, or (f) analkoxyl; R³ is (a) hydrogen, (b) an alkyl having 1 to 4 carbon atomswhich may optionally be substituted with an alicyclic group, or (c) analkoxyl; R⁴ is a substituted or unsubstituted benzyl; R⁵ is (a) hydrogenor (b) a substituted or unsubstituted alkyl; X¹ is (a) hydrogen or (b)hydroxy; and, X² is (a) hydrogen or (b) hydroxy; with the proviso thatwhen R² and R³ are hydrogen, R¹ is (a) an alkyl having 1 to 4 carbonatoms which may optionally be substituted with an alicyclic group or (b)trifluoromethyl, when R¹ and R³ are hydrogen, R² is (a) hydroxy, (b) analkyl having 1 to 4 carbon atoms which may optionally be substitutedwith an alicyclic group, (c) trifluoromethyl, or (d) an alkoxyl, and,when R¹ and R² are hydrogen, R³ is (a) an alkyl having 1 to 4 carbonatoms which may optionally be substituted with an alicyclic group or (b)an alkoxyl.
 35. The method according to claim 34, wherein R¹ ishydrogen, hydroxy, methyl, ethyl, propyl, adamantylmethyl,cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, ortrifluoromethyl, in the general formula (1).
 36. The method according toclaim 34, wherein R² is hydrogen, hydroxy, fluorine, methyl, ethyl,propyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl,cyclohexylethyl, trifluoromethyl, methoxy, ethoxy, n-propoxy,iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy, ortert-butoxy, in the general formula (1).
 37. The method according toclaim 34, wherein R³ is hydrogen, methyl, ethyl, propyl,adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl,methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy,iso-butoxy, sec-butoxy, or tert-butoxy, in the general formula (1). 38.The method according to claim 34, wherein R⁴ is benzyl in the generalformula (1).
 39. The method according to claim 34, wherein, in thegeneral formula (1): R¹ is hydrogen, methyl, ethyl, or trifluoromethyl;R² is hydrogen, hydroxy, methyl, or methoxy; R³ is hydrogen or methyl;R⁴ is benzyl; R⁵ is hydrogen; X¹ is hydroxy; and, X² is hydrogen; withthe proviso that when R² and R³ are hydrogen, R¹ is methyl, ethyl, ortrifluoromethyl, when R¹ and R³ are hydrogen, R² is hydroxy, methyl, ormethoxy, and, when R¹ and R² are hydrogen, R³ is methyl.
 40. The methodaccording to claim 39, wherein the compound is represented by formulabelow:


41. The method according to claim 39, wherein the compound isrepresented by formula below:


42. The method according to claim 39, wherein the compound isrepresented by formula below:


43. The method according to claim 39, wherein the compound isrepresented by formula below:


44. The method according to claim 39, wherein the compound isrepresented by formula below:


45. The method according to claim 39, wherein the compound isrepresented by formula below:


46. The method according to claim 39, wherein the compound isrepresented by formula below:


47. The method according to claim 34, wherein Renilla sp. is Renillareniformis.
 48. The method according to claim 47, wherein the luciferasederived from Renilla reniformis comprises a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:
 14. 49. The method according toclaim 34, wherein Oplophorus sp. is Oplophorus gracilorostris.
 50. Themethod according to claim 49, wherein the luciferase derived fromOplophorus gracilorostris comprises a polypeptide consisting of theamino acid sequence of SEQ ID NO:
 16. 51. The method according to claim34, wherein Gaussia sp. is Gaussia princeps.
 52. The method according toclaim 51, wherein the luciferase derived from Gaussia princeps comprisesa polypeptide consisting of the amino acid sequence of SEQ ID NO: 18.